Fermentation processes for cultivating streptococci and purification processes for obtaining cps therefrom

ABSTRACT

This invention is in the field of bacterial cultures and specifically relates to the optimization of culture conditions to improve the production of bacterial capsular polysaccharides from  Streptococcus  strains in fed batch culture and to novel purification methods suitable for production scale purification of bacterial capsular polysaccharides from  Streptococcus  strains resulting in higher levels of purity than previously obtained for production scale.

FIELD OF THE INVENTION

This invention is in the field of bacterial cultures, and preferablyrelates to the optimization of culture conditions and novel purificationmethods to improve the production of bacterial capsular polysaccharides.

BACKGROUND OF THE INVENTION

Capsular polysaccharides (cps) are important immunogens found on thesurface of bacteria involved in various bacterial diseases. This featurehas led to them being an important component in the design of vaccines.They have proved useful in eliciting immune responses especially whenlinked to carrier proteins (Ref 1).

Typically, capsular polysaccharides are produced using batch culture incomplex medium (Group B Streptococcus, Staphylococcus aureus,Streptococcus pneumoniae and Haemophilus influenzae), fed batch culture(H. influenzae) or continuous culture (Group B Streptococcus andLactobacillus rhamnosus) (Refs. 2-7). Most studies used batch culturesystems in which the growth rate, nutrient levels and metabolicconcentrations change during incubation. In such systems, alteration ofone factor results in changes in other factors associated with growththat can affect yields unpredictably. Continuous cultures allow theresearcher to separate and define parameters that are interdependentduring batch culture growth, such as growth rate, nutrient and productconcentrations and cell density. During continuous culture, fresh mediumis added to a culture at a fixed rate and cells and medium are removedat a rate that maintains a constant culture volume. Continuous culturewas preferred for capsular polysaccharide production when it proved tobe dependent on conditions (Ref. 8).

For Group B Streptococcus (GBS, S. agalactiae), cell growth rate wasreported to be the principal factor regulating capsular polysaccharideproduction. Furthermore, the production of type III capsularpolysaccharide was shown to occur independently of the growth-limitingnutrient. Higher specific yields (up to about 90 mg/g_(CWD)) wereobtained when cells were held at a fast (0.8, 1.4 or 1.6 h) massdoubling time (t_(d)) rather than at a slow time (t_(d)=2.6 or 11 h)(Refs. 8-10). However, continuous culture is prone to strain stabilityproblems and contamination, and is somewhat expensive due to thecontinuous feed of medium and nutrients. Therefore, there is a need tofind alternatives to continuous culture for the high yield production ofcapsular polysaccharides in order to overcome the problems withcontinuous culture that are cited above.

One approach to overcome the drawbacks of continuous culture isexemplified in WO 2007/052168. A complex fed batch fermentation processhas been developed to maintain a nutritional environment and a growthrate favorable to cps production. This process combines the advantagesof batch and continuous techniques, producing high cell densities due toextension of the exponential growth phase and to conditions that controlsubstrate addition during fermentation. However, the complex fed batchtechnique uses software with a complex algorithm to manage thefermentation. Furthermore, a robust and cost-effective productionprocess in compliance with Good Manufacturing Practices is necessary togenerate material to support clinical trials. Therefore, there is anurgent need to simplify the fed batch fermentation process forlarge-scale production.

In addition to a need for simplified fermentation protocols, there is aneed for simplified purification protocols that can be used in thelarge-scale production of capsular polysaccharides post-fermentation.The approach exemplified in WO 2007/052168 is based on the methoddisclosed in WO 2006/082527, which includes extraction, alcoholicprecipitation, diafiltration, cationic detergent treatment, andre-solubilization. This procedure is highly efficient and typicallyyields a preparation of capsular polysaccharide that is approximately80% pure. However, the step of cationic detergent treatment results inprecipitation of the capsular polysaccharide. The subsequent separationof the precipitate from the supernatant (e.g. by centrifugation) andre-solubilization is laborious and may result in loss of capsularpolysaccharide, thereby reducing yield. The efficiency of the cationicdetergent treatment may also be dependent on the initial purity of thecapsular polysaccharide. The lower the initial purity of the capsularpolysaccharide, the less efficient the cationic detergent treatment maybe, further limiting yield. Therefore, there is a need for a simplifiedpurification procedure that will produce higher levels of purity withfewer complicated and/or expensive purification steps. There is also aneed for a purification procedure that provides a good yield of capsularpolysaccharide whatever the initial purity of the polysaccharide.

SUMMARY OF THE DISCLOSED EMBODIMENTS

The inventors have met the need for simplified fermentation protocols byproviding methods for producing capsular polysaccharides (cps) fromStreptococcus on a manufacturing scale. In certain embodiments, thealgorithm for pH balancing during linear addition of a carbon source hadbeen eliminated, and in other embodiments, unnecessary components of themedia have been omitted. The preferred species of Streptococcus isStreptococcus agolactiae, also referred to as Lancefield's Group BStreptococcus or GBS, in particular, strains O90, 1-136b, CJB111, orM781.

One aspect provides an inoculum of a strain of Streptococcus thatexpresses cps. In one embodiment, the optical density (OD) of theinoculum is preferably between 0.6-1.8, which is the mid-exponentialphase of the inoculum. Although the reported OD values are measured at590 nm, OD can be converted based on the absorbance wavelength of agiven experiment.

Another aspect provides a method for cultivating the Streptococcusstrain by fermentation. In one embodiment, the pH of the cultivatingmedium during the cultivating is between 6.0-7.5, preferably about 7.3.In another embodiment, the temperature of the cultivating medium duringthe cultivating is between 34-38° C., preferably about 36° C.

Another aspect provides a method for cultivating the Streptococcusstrain, wherein the cultivating comprises two instantaneous additions ofyeast extract, followed by a linear addition of a carbon source. Thepreferred carbon source for the linear addition is glucose. Eachaddition is initiated at a designated OD level, which has been selectedto achieve a higher volumetric production of cps by regulating thebacteria growth rate and to adapt the micro-organism to produce amaximum serotype specific cps.

In one embodiment, the first instantaneous addition of yeast extract isinitiated at an OD level between 2.8-3.2, preferably about 3.0. Inanother embodiment, the second instantaneous addition of yeast extractis initiated at an OD level between 4.3-4.7, preferably about 4.5. Inanother embodiment, the linear addition of the carbon source isinitiated at an OD level between 9.8-10.0, preferably about 10.

Overall, the linear addition of a carbon source without an algorithm isan improvement over the previous complex fed batch fermentation processthat used an algorithm to control the cultivating by monitoring a pH ofthe cultivating medium.

Another aspect provides a cultivating medium that includes a definedmedium or a complex medium. The defined medium comprises a phosphatesource, a mineral source, a carbon source, a vitamin source, and anamino acid source to grow Streptococcus. The vitamin source consists ofsix or fewer vitamins selected from the following list of sevenvitamins: biotin, niacinamide, calcium pantothenate, riboflavin,thiamine hydrochloride, pyridoxine hydrochloride and folic acid, whereintwo of the vitamins have to be calcium pantothenate and niacinamide.

The complex medium comprises a complex extract (preferably yeastextract), a phosphate source, a carbon source, a vitamin source, andoptionally an amino acid source to grow Streptococcus. The vitaminsource consists of four or fewer vitamins selected from the followinglist of five vitamins: biotin, niacinamide, riboflavin, thiaminehydrochloride and pyridoxine hydrochloride, wherein one of the vitaminshas to be biotin (i.e., four of the five are included in the mediumwhile the fifth is not added other that as a natural component of thecomplex extract). In preferred embodiment, the vitamin source has threeor fewer, two or fewer, or biotin only.

The invention further provides a composition including a cultivatingmedium that is a defined medium, which is comprised of a phosphatesource, a mineral source, a carbon source, a vitamin source, and anamino acid source to grow Streptococcus. Once again, the preferredstrain of Streptococcus is Streptococcus agalacticte, in particular,strain O90, H36b, CJB111, or M781. In one embodiment, the phosphatesource consists of K₂HPO₄, KH₂PO₄, Na₂HPO₄.H₂O, NaH₂PO₄.H₂O, or NaCl. Inone embodiment, the preferred carbon source is glucose.

In another embodiment, the vitamin source consists of six or fewervitamins selected from the following list of seven vitamins: biotin,niacinamide, calcium pantothenate, riboflavin, thiamine hydrochloride,pyridoxine hydrochloride and folic acid, wherein two of the vitaminshave to be calcium pantothenate and niacinamide.

In another embodiment, the vitamin source consists of five or fewer fromthe following list of seven vitamins: biotin, niacinamide, calciumpantothenate, riboflavin, thiamine hydrochloride, pyridoxinehydrochloride, and folic acid, wherein two have to be calciumpantothenate and niacinamide.

In another embodiment, the vitamin source consists of four or fewer fromthe following list of seven vitamins: biotin, niacinamide, calciumpantothenate, riboflavin, thiamine hydrochloride, pyridoxinehydrochloride, and folic acid, wherein two have to be calciumpantothenate and niacinamide.

In another embodiment, the vitamin source consists of three or fewerfrom the following list of seven vitamins: biotin, niacinamide, calciumpantothenate, riboflavin, thiamine hydrochloride, pyridoxinehydrochloride, and folic acid, wherein two have to be calciumpantothenate and niacinamide.

In another embodiment, the vitamin source consists of calciumpantothenate and niacinamide.

In another embodiment, the amino acid source consists of nineteen orfewer from the following list of nineteen amino acids: alanine,arginine, glutamine, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,valine, aspartic acid, cysteine hydrochloride, glutamic acid, andtyrosine, wherein fifteen have to be arginine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, serine,threonine, tryptophan, valine, cysteine hydrochloride, glutamic acid,and tyrosine.

In another embodiment, the amino acid source consists of eighteen orfewer from the following list of nineteen amino acids: alanine,arginine, glutamine, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,valine, aspartic acid, cysteine hydrochloride, glutamic acid, andtyrosine, wherein fifteen have to be arginine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, serine,threonine, tryptophan, valine, cysteine hydrochloride, glutamic acid,and tyrosine.

In another embodiment, the amino acid source consists of seventeen orfewer from the following list of nineteen amino acids: alanine,arginine, glutamine, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,valine, aspartic acid, cysteine hydrochloride, glutamic acid, andtyrosine, wherein fifteen have to be arginine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, serine,threonine, tryptophan, valine, cysteine hydrochloride, glutamic acid,and tyrosine.

In another embodiment, the amino acid source consists of sixteen orfewer from the following list of nineteen amino acids: alanine,arginine, glutamine, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tiyptophan,valine, aspartic acid, cysteine hydrochloride, glutamic acid, andtyrosine, wherein fifteen have to be arginine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, serine,threonine, tryptophan, valine, cysteine hydrochloride, glutamic acid,and tyrosine.

In another embodiment, the amino acid source consists of arginine,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, serine, threonine, tryptophan, valine, cysteinehydrochloride, glutamic acid, and tyrosine.

Another aspect provides a composition including a cultivating mediumthat is a complex medium, which is comprised of a complex extract(preferably a yeast extract), a phosphate source, a carbon source, avitamin source, and optionally an amino acid source to growStreptococcus. Once again, the preferred strain of Streptococcus isStreptococcus agalactiae, in particular, strain O90, H36b, CJB111, orM781.

In one embodiment, the vitamin source consists of four or fewer vitaminsselected from the following list of five vitamins: biotin, niacinamide,riboflavin, thiamine hydrochloride and pyridoxine hydrochloride, whereinone of the vitamins has to be biotin.

In another embodiment, the vitamin source consists of three or fewervitamins selected from the following list of five vitamins: biotin,niacinamide, riboflavin, thiamine hydrochloride and pyridoxinehydrochloride, wherein one of the vitamins has to be biotin.

In another embodiment, the vitamin source consists of two or fewervitamins selected from the following list of five vitamins: biotin,niacinamide, riboflavin, thiamine hydrochloride and pyridoxinehydrochloride, wherein one of the vitamins has to be biotin.

In another embodiment, the vitamin source is biotin.

The foregoing aspects and embodiments are not intended to be exclusiveof one another and may be combined with each other and any other aspectsor embodiments disclose in this specification except to the extendmutually exclusive.

The invention further provides a method for purifying a capsularpolysaccharide, typically from Streptococcus agalactiae, comprising astep of filtration using an adherent filter. The adherent filter is onethat binds contaminants that may be present in the capsularpolysaccharide, e.g. proteins and/or nucleic acids, while allowing thecapsular polysaccharide to pass through the filter. The inventors havefound that adherent filters can be used to purify capsularpolysaccharides instead of the cationic detergent treatment described inWO 2007/052168 and WO 2006/082527. The use of an adherent filter removesthe need to apply a cationic detergent, which means that there is noprecipitation of the capsular polysaccharide at this stage of themethod. This in turn removes the need to separate the precipitate fromthe supernatant, simplifying the method and preventing any loss of thecapsular polysaccharide that may occur during this separation. The useof an adherent filter can therefore improve the yield of thepurification method. The efficiency of the adherent filter is also lessdependent on the initial purity of the capsular polysaccharide.

The skilled person is capable of identifying suitable adherent filtersfor use in this method. Typically, the main contaminant in the capsularpolysaccharide is protein, and the adherent filter is therefore aprotein adherent filler. The inventors have found that carbon filtersare particularly suitable. They typically comprise activated carbon(e.g. as a granular carbon bed or as a pressed or extruded carbonblock), which acts as the filter for purification of the sample.

The skilled person is capable of identifying suitable carbon filters.Typically, a carbon filter for use in the present invention containsactivated carbon immobilized in a matrix. The matrix may be any porousfilter medium permeable for the sample. The matrix may comprise asupport material and/or a binder material. The support material may be asynthetic polymer or a polymer of natural origin. Suitable syntheticpolymers may include polystyrene, polyacrylamide and polymethylmethacrylate, while polymers of natural origin may include cellulose,polysaccharide and dextran, agarose. Typically, the polymer supportmaterial is in the form of a fibre network to provide, mechanicalrigidity. The binder material may be a resin. The matrix may have theform of a membrane sheet. Typically, the activated carbon immobilized inthe matrix may be in the form of a cartridge. A cartridge is aself-contained entity containing powdered activated carbon immobilizedin the matrix and prepared in the form of a membrane sheet. The membranesheet may be captured in a plastic permeable support to form a disc.Alternatively, the membrane sheet may be spirally wound. To increasefilter surface area, several discs may be stacked upon each other. Inparticular, the discs stacked upon each other have a central core pipefor collecting and removing the carbon-treated sample from the filter.The configuration of stacked discs may be lenticular. The activatedcarbon in the carbon filter may be derived from different raw materials.e.g. peat, lignite, wood or coconut shell. Any process known in the art,such as steam or chemical treatment, may be used to activate carbon. Inthe present invention, activated carbon immobilized in a matrix may beplaced in a housing to form an independent filter unit. Each filter unithas its own in-let and out-let for the sample to be purified. Examplesof filter units that are usable in the present invention are the carboncartridges from Cuno Inc. (Meriden, USA) or Pall Corporation (East Hill,USA).

In particular, the inventors have found that CUNO Zetacarbon™ filtersare suitable for use in the invention. These carbon filters comprise acellulose matrix into which activated carbon powder is entrapped andresin-bonded in place.

The starting material for the method of this aspect of the invention maybe one of the starting materials described in the section entitled“Starting material” below. The method may additionally comprise one ormore of the steps described in the sections entitled “Alcoholicprecipitation and cation exchange”, “Diafiltration”, “Re-N-acetylation”,“Further diafiltration”, “Conjugate preparation” and/or “Other steps”below. A typical sequence of steps would therefore be i) a step or stepsdescribed in the section entitled “Alcoholic precipitation and cationexchange”; ii) a step or steps described in the section “Diafiltration”;iii) a step of filtration using an adherent filter, as described above;iv) a step or steps described in the section entitled“Re-N-acetylation”; and v) a step or steps described in the sectionentitled “Further diafiltration”. This process may then be followed by astep or steps described in the section entitled “Conjugate preparation”.Finally, this process may be followed by a step or steps described inthe section entitled “Other steps”.

The method may additionally comprise one or more of the steps describedin the sections entitled “Cationic detergent treatment” and“Re-solubilization” below, although typically these steps are omittedbecause cationic detergent treatment to precipitate the capsularpolysaccharide and subsequent re-solubilization of the polysaccharide isgenerally not required when filtration is carried out using an adherentfilter in the method of the invention. Accordingly, the inventionspecifically envisages a method for purifying a capsular polysaccharide,typically from Streptococcus agalactiae, comprising a step of filtrationusing an adherent filter, wherein the method does not include a step ofcationic detergent treatment to precipitate the capsular polysaccharidefollowed by a step of re-solubilization of the capsular polysaccharide.

The invention further provides methods for purifying capsularpolysaccharides (cps) from Streptococcus also on a manufacturing scale.The preferred species of Streptococcus is Streptococcus agalactiae, alsoreferred to as Lancefield's Group B Streptococcus or GBS, in particular,strains O90, H36b, CJB111, or M781.

In a preferred embodiment the method for production of a purifiedcapsular polysaccharide includes one or more of the following steps: (a)providing a crude isolate containing a capsular polysaccharide; (b)removing an alcohol precipitate formed by contacting the crude isolatewith an alcohol solution; (c) filtering to remove smaller molecularweight compounds while retaining the capsular polysaccharide; and (d)removing protein contaminants with a protein adherent filter to producethe purified capsular polysaccharide. In a preferred embodiment, themethod includes all of the foregoing steps. In a more preferredembodiment, the method omits detergent precipitation.

In certain embodiments, one or more additional steps may be performedincluding (e) re-N-acetylating the purified capsular polysaccharide, (f)precipitating the purified capsular polysaccharide; and/or (g)formulating a vaccine with the capsular polysaccharide as a component.

In certain embodiments the alcohol solution added to a concentrationsufficient to precipitate nucleic acid contaminants but not the capsularpolysaccharide. In preferred embodiments, the alcohol is ethanolpreferably added to a concentration of between about 10% and about 50%ethanol, more preferably to a concentration of between about 30%ethanol. The alcohol solution may optionally include a cation,preferably a metal cation, more preferably a divalent cation, mostpreferably calcium.

In certain embodiments, the protein adherent filter is an activatedcarbon filter.

BRIEF DESCRIPTION THE FIGURES

FIG. 1 shows the capsular polysaccharides that are potential GBS vaccinetargets.

FIG. 2 is the schematic representation of a proposed model for thelinkage of capsular polysaccharides (cps) and Group B carbohydrate ofGBS.

FIG. 3A shows the molecular structure of serotype specific cps of GBSfrom Type Ia and Type Ib.

FIG. 3B shows the molecular structure of serotype specific cps of GBSfrom Type III and Type V.

FIG. 4 shows the production process of glycoconjugate vaccine againstGBS.

FIGS. 5A-D shows the growth curve and pH of medium for the (A) O90strain, (B) H36b strain, (C) M781 strain, and (D) CJB111. The specificgrowth rate was calculated using OD values in a range of 0.1-2.4.

FIG. 6 shows (A) the growth curve for the O90 strain. (B) the cpsconcentration by the O90 strain, and (C) the cps production by gram celldry weight, wherein the growth rate was determined for biotin and fourvitamins in sodium hydroxide and methanol; biotin and three vitamins inwater without riboflavin; only biotin: and without vitamins.

FIG. 7 shows (A) the growth curve for the H36b strain, (B) the cpsconcentration by the H36b strain, and (C) the cps production by gramcell dry weight, wherein the growth rate was determined for biotin andfour vitamins in sodium hydroxide and methanol: biotin and threevitamins in water without riboflavin; and only biotin.

FIG. 8 shows (A) the growth curve for the M781 strain, (B) the cpsconcentration by the M781 strain, and (C) the cps production by gramcell dry weight, wherein the growth rate was determined for biotin andfour vitamins in sodium hydroxide and methanol; biotin and threevitamins in water without riboflavin; and only biotin.

FIG. 9 shows the growth curve for the CJB111 strain, the cpsconcentration by the CJB111 strain, and the cps production by gram celldry weight, wherein the growth rate was determined for biotin alone.

FIG. 10 shows (A) the growth curve for the O90 strain, (B) the cpsconcentration by the O90 strain, and (C) the cps production by gram celldry weight, wherein the growth rate was determined for the feed batchtechnique; the instantaneous addition of yeast extract when the OD levelis at 3 and at 5, followed by a linear addition of glucose; and theaddition of the entire yeast extract in batch medium, followed by alinear addition of glucose.

FIG. 11 shows (A) the growth curve for the H36b strain, (B) the cpsconcentration by the H36b strain, and (C) the cps production by gramcell dry weight, wherein the growth rate was determined for the feedbatch technique; the instantaneous addition of yeast extract when the ODlevel is at 3 and at 5, followed by a linear addition of glucose; andthe addition of the entire yeast extract in batch medium, followed by alinear addition of glucose.

FIG. 12 shows (A) the growth curve for the M781 strain, (B) the cpsconcentration by the M781 strain, and (C) the cps production by gramcell dry weight, wherein the growth rate was determined for the feedbatch technique: the instantaneous addition of yeast extract when the ODlevel is at 3 and at 5, followed by a linear addition of glucose; andthe addition of the entire yeast extract in batch medium followed by alinear addition of glucose.

FIG. 13 shows the growth curve for the CJB111 strain, the cpsconcentration by the CJB111 strain, and the cps production by gram celldry weight, wherein the growth rate was determined for the instantaneousaddition of yeast extract when the OD level is at 3 and at 5, followedby a linear addition of glucose.

FIG. 14 provides the results of a DOT study, which shows (A) the growthcurve for the H36b strain at 15%, 30%, and 60%, (B) the cpsconcentration by H36b strain, (C) the cps production by gram cell dryweight, and (D) the average productivity of the H36b strain.

FIG. 15 provides the results of a temperature study, which shows (A) thegrowth curve for the H36b strain at 34° C., 36° C., and 38° C., (B) thecps concentration by H36b strain, (C) the cps production by gram CELLDRY WEIGHT, and (D) the average productivity of the H36b strain.

FIG. 16 provides the results of a pH study, which shows (A) the growthcurve for the H36b strain at 7.0, 7.3, and 7.5, (B) the cpsconcentration by H36b strain, (C) the cps production by gram cell dryweight, and (D) the average productivity of the H36b strain.

FIG. 17 provides the results of a pressure study, which shows (A) thegrowth curve for the H36b strain at 0.2 and 0.5 bar, (B) the cpsconcentration by H36b strain, (C) the cps production by gram cell dryweight, and (D) the average productivity of the H36b strain.

FIG. 18 shows the growth curve for M781 strain in (A) 500 mL Erlenmeyerflasks containing 100 mL of chemically defined medium (0.1 mL of w.s.),and (B) 2 L fermentor. The specific growth rate was calculated using ODvalues in a range of 0.1-0.7.

FIG. 19 shows the growth curve for the M781 strain as a plot graph, andglucose consumption as a bar graph for the standard batch medium; 10times the quantity of vitamins; 10 times the quantity of amino acids andvitamins; and 10 times the quantity of vitamins. amino acids andpotassium.

FIG. 20 shows the effect of the omission of alanine, aspartic acid,glutamine and proline on the growth of strain M781 of GBS in a definedmedium.

FIG. 21 shows the effect of omission of biotin, folic acid, pyridoxine,riboflavin and thiamine on the growth of strain M781 of GBS in a definedmedium.

FIG. 22 shows the OD_(590nm) profile of the first pre-test fermentationruns of the 3 strains, M781, H36b and O90, at a laboratory-scale.

FIG. 23 shows the OD_(590nm) profile of the second pre-test fermentationruns of the 4 strains, M781, H36b, O90, and CJB111, using a simplifiedprocess. The first simplification removed thiamine, riboflavin,pyridoxine HCl, and niacinamide from the vitamin solution. The secondsimplification was the modification of the parameters of the fed phasesduring the fermentation.

FIG. 24 shows the OD_(590nm) profile of the test fermentation runs ofthe 4 strains, M781, H36b, O90, CJB111.

FIG. 25 shows the ¹H NMR spectrum of purified GBS Type Ia polysacchariderecorded at 25° C. Certain hydrogen are identified on the spectrum.

FIG. 26 shows the ¹H NMR spectrum of purified GBS Type Ib polysacchariderecorded at 25° C. Certain hydrogen are identified on the spectrum.

FIG. 27 shows the ¹H NMR spectrum of purified GBS Type IIIpolysaccharide recorded at 25° C. Certain hydrogen are identified on thespectrum.

FIG. 28 shows the ¹H NMR spectrum of purified GBS Type V polysacchariderecorded at 25° C. Certain hydrogen are identified on the spectrum.

FIG. 29 shows an overlay of elution profiles of a polysaccharide sampleand sialic acid standard (gray line) at 0.5 μg/ml.

FIG. 30 shows an overlay of elution profiles of a polysaccharide sampleand a polysaccharide sample with rhamnose added (gray line).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The inventors have discovered that high yields of cps on a manufacturingscale can be obtained for any Streptococcus strain using fed batchculture, that is a culture which is initiated by the inoculation ofcells into a finite volume of fresh medium and terminated by a singleharvest after the cells have grown, with extra nutrients being added tothe culture once the initial source of nutrients has been exhausted.Such high yields are comparable to or better than those obtained usingcontinuous culture. Furthermore, the methods disclosed herein are notprone to the stability and contamination problems of continuous culture.The inventors have further developed an optimized purification protocolwhich significantly improves the impurities while keeping the protocolsimple and inexpensive for manufacturing scale.

This disclosure provides a process for culturing Streptococcus, whereinthe Streptococcus is grown in fed batch culture. Certain strains ofStreptococcus are known to be “bad producers” of cps in that theytypically produce low levels of cps in a culture. Examples of such badproducers include the GBS strains DK21 and 2603. However, using themethods disclosed herein, high levels of cps can be obtained even fromsuch strains that are known to produce lower levels of cps. Thereforethe invention provides a process for increasing the cps yield from astrain of Streptococcus comprising culturing Streptococcus in fed batchculture wherein, under batch or continuous culture conditions, thestrain would only produce <30 mg cps/g_(CDW) or <10 mg cps/g_(CDW) inthe case of “bad producers.”

Preferably the invention provides a method of culturing Streptococcus infed batch culture, wherein a high yield of cps is produced. Preferablythe yield of cps is 10 mg/g_(CDW) or more in the case of bad producers(preferably 15, 20, 25, or 30 or more) and 30 mg/g_(CDW) or more in thecase of other strains (preferably 40, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200 or more). Preferably the yield ofcps from the culture medium is 10 mg/L or more (e.g., 20, 30, 40, 50,60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300 ormore). More preferably, the yield of cps from the culture medium is 50mg/L or more (e.g., 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650 or more). Thus, this invention allows the production of cps at a farhigher yield per unit volume compared with continuous culture. In somecases, the yield per unit volume may be at least twice the quantityproduced using continuous culture, more preferably two and one halftimes or three times the quantity produced using continuous culture.

This invention also provides a method for cultivating the Streptococcusstrain by fermentation, comprising two instantaneous additions of yeastextract, followed by a linear addition of a carbon source, preferablywithout use of an algorithm to monitor pH. The preferred carbon sourcefor the linear addition is glucose. Each addition is initiated at adesignated OD level, which is selected to achieve a higher volumetricproduction of cps by regulating the bacteria growth rate and to adaptthe micro-organism to produce a maximum serotype specific cps.

In one embodiment, the first instantaneous addition of yeast extract isinitiated at an OD level between 2.8-3.2, preferably about 3.0. Inanother embodiment, the second instantaneous addition of yeast extractis initiated at an OD level between 4.3-4.7, preferably about 4.5. Inanother embodiment, the linear addition of the carbon source isinitiated at an OD level between 9.8-10.0, preferably about 10.

Overall, the linear addition of a carbon source is an improvement overthe previous complex fed batch fermentation process that used analgorithm to control the cultivating by monitoring a pH of thecultivating medium.

Following the cultivating, the bacteria may undergo further processingsteps in order to purify the cps and to conjugate it to a carrierprotein. The invention therefore may further comprise steps of purifyingcps from the bacteria, and conjugating the capsular saccharide to acarrier protein, to give a protein-saccharide conjugate (see FIGS. 1-2).The purified cps may undergo further processing steps in order toprepare pharmaceutical preparations. In preferred embodiments, thepurification will be carried out using the improved purificationprotocol disclosed herein.

Streptococcus

The term “Streptococcus” refers to bacteria that may be selected from S.agalactiae (GBS), S. pyogenes (GAS), S. pneumoniae (pneumococcus) and S.mutans. The streptococcus may alternatively be S. thermophilus or S.lactis. Preferably the Streptococcus is GBS. If the Streptococcus usedis GBS, then preferably the serotype selected is 1a, 1b, 3, 4 or 5.Preferably the strains of GBS used are O90 (1a), 7357 (1b), H36b (1b),DK21 (2), M781 (3), 2603 (5), or CJB111 (5). See FIGS. 3A-B. If theStreptococcus used is S. pneumoniae, then preferably the serotypesselected are one or more, or all of 4, 6B, 9V, 14, 18C, 19F, and 23F.Serotype 1 may also preferably be selected. Preferably the serotypesselected are one or more, or all of 1, 3, 4, 5, 6B, 7F, 9V. 14, 18C,19F, and 23F.

Moreover, the culture produced using the method of the invention may behomogeneous (i.e. consists of a single species or strain ofStreptococcus), or may be heterogeneous (i.e. comprises two or morespecies or strains of Streptococcus). Preferably the culture ishomogeneous.

The Streptococcus used may be a wild type strain or may be geneticallymodified. For instance, it may be modified to produce non-naturalcapsular polysaccharides or heterologous polysaccharides or to increaseyield.

Production Process Overview

The production of GBS can be divided into four parts: (1) the productionby fermentation of each of the cps and their primary recovery: (2) thepurification of the microfiltration permeate; (3) the formulation of thedried purified cps; and (4) the characterization of the glycoconjugatebiomolecules.

The first step may be optimized in a pilot-scale fermentation hall, andconsists of the production of biomass by fermentation, the continuousflow centrifugation of the biomass, the collection of the pellet (orcellular paste), the inactivation of the microorganism and the releaseof the cps, and finally the microfiltration of the cellular paste withthe collected permeate. The fermentation consists of (1) inoculumpreparation, (2) the fermentation, the centrifugation of the biomass,the chemical treatments of the pellet and the microfiltration of thepellet as presented in FIG. 4.

The invention provides a method for producing cps on a manufacturingscale, which includes a method for providing an inoculum of a strain ofStreptococcus expressing the cps, and a method for cultivating thestrain by fermentation. The cultivating consists of monitoring theoptical density (OD) of the cultivating medium such that when the ODreaches designated addition levels which prompts the two instantaneousadditions of yeast extract, followed by a linear addition of a carbonsource to a cultivating medium as opposed to an algorithm to control thecultivating by monitoring a pH of the cultivating medium.

Culture of the Inoculum

The culture of the inoculum may be performed in shake flasks sterilizedusing an autoclave at 121° C. The inoculum contains complex medium(consisting of yeast extract, Na₂HPO₄.2H₂O, NaH₂PO₄.H₂O, andmonohydrated glucose with a neutral pH approximately 7.3), a solution ofvitamins (consisting of thiamine, riboflavin, pyridoxine HCl, andniacinamide, diluted in NaOH), and a biotin solution. In preferredembodiments, the solution of vitamins is omitted and only the biotinsolution is used as a vitamin supplement.

In a preferred embodiment, each flask is inoculated with 2.75±0.25 mL ofworking seeds. The culture is maintained at approximately 35° C. withagitation at approximately 200 rpm in the incubator for approximately 4hours. After this time, the biomass concentration was evaluated bymeasuring the OD at 590 nm and performing a Gram stain. If the value ofOD_(590nm) is between approximately 0.6-1.8, and if the Gram stainproduces only Gram positive cocci, the contents of the flasks are pooledinto a heat-sterilized bottle connected to the incubation line of thefermentor.

During the inoculum preparation, the preferred conditions are asfollows: the initial pH of the medium is 7.3±0.1, the volume of workingseed is 2.5-3.0 ml/flask, the temperature of incubation is 35±1° C., andthe agitation speed is 200±10 rpm. At the end of the culture in theflasks, the preferred final OD_(590nm) is between 0.6-1.8, and thepreferred Gram stain produces only Gram positive cocci. In the pooledbottle, the preferred purity of the culture is such that there is nocontaminant. Finally, the preferred time of incubation is between 3-5hours.

Fed Batch Fermentation Process

The invention provides an improved method of culturing the Streptococcususing a fed batch process on a manufacturing scale (see FIGS. 6-18). Fedbatch culture may be either fixed volume fed batch or variable volumefed batch. In fixed volume fed batch culture, the limiting substrate isfed without diluting the culture (e.g., using a concentrated liquid orgas or by using dialysis). In variable volume fed batch culture, thevolume changes over fermentation time due to the substrate feed.

During the fermentation process in the 300 L fermentor, the preferredconditions are as follows: the temperature of the culture is set at36±1° C., the overpressure inside the fermentor is set at approximately0.2 bar, the pH is set at 7.3±0.1 and adjusted using 4 M NaOH, theinitial stir is set at 50 rpm, the initial airflow is set at 20 L/min,the level of foam in the fermentor is visually monitored and adjustedusing antifoam PPG 2500 if necessary, the dissolved oxygen tension (DOT)is set at 300/o and regulated in cascade by stirring (between 50-350rpm), the air airflow (between 20-100 L/min, and the oxygen flow(between 0-100 L/min).

This invention provides two instantaneous additions of yeast extract atspecified OD levels, followed by a linear addition of a carbon source tothe cultivating medium. Samples are taken during the batch phase of thefermentation, two hours after inoculation, and the OD_(590nm) ismeasured. Samples are taken every 15 minutes until the OD_(590nm)reaches 3 at which point the first instantaneous batch addition isinitiated using a 150 g/L yeast extract solution. Approximately 45minutes after the first addition, the OD_(590nm) is measured again.Samples are taken every 15 minutes until the OD_(590nm) reaches 5, atwhich point a second instantaneous batch addition is initiated using a150 g/L yeast extract solution. When the OD_(590nm) reaches 10-12, alinear addition is initiated. During this linear addition, a sample istaken every hour to measure the OD_(590nm). The linear addition lastsapproximately 3 hours at which time the automatic controls of theparameters are stopped. The stir is regulated at 100 rpm and thetemperature at 30° C.

Growth Medium

Any type of liquid growth medium may be used which is suitable formaintaining growth of Streptococcus species. Preferred media includecomplex media such as Columbia broth, LB, Todd-Hewitt, OC medium, bloodbroth or brain-heart infusion: semi-defined media such as MCDM:chemically defined media for Streptococcus such as MI, MC, FMC (Ref 11),or C-48 (Ref 12); and media composed for growth of eukaryotic cell linescontaining necessary auxotrophic components such as RPMI, spent medium,McCoy's and Eagle's. A typical growth medium contains yeast extract, aswell as other factors essential for growth including lipids (long chainfatty acids such as linoleic or oleic acid), steroids (such ascholesterol), purines and pyrimidines, minerals, vitamins and growthfactors, amino acids (L- and/or D-form) and/or chemical elements orinorganic ions (such as Fe, K, Mg, Mn, Ca. Co, Cu, P and/or Zn). Byincreasing the concentration of the medium, higher ODs may be achieved,resulting in a higher volumetric production of cps. Accordingly, thecomplex medium preferably comprises yeast extract, a phosphate source, acarbon source, a vitamin source, and optionally an amino acid source togrow Streptococcus, wherein the vitamin source consists of biotin, andoptionally one or more vitamins chosen from niacinamide, riboflavin,thiamine hydrochloride and pyridoxine hydrochloride.

The chemically defined medium preferably comprises a phosphate source, amineral source, a carbon source, a vitamin source, and an amino acidsource to grow Streptococcus, wherein the vitamin source consists ofcalcium pantothenate, niacinamide, and one or more vitamins chosen frombiotin, riboflavin, thiamine hydrochloride, pyridoxine hydrochloride andfolic acid.

The growth medium may additionally comprise one or more of an antibioticand an antifoam agent. Typical antibiotics include kanamycin, ampicillinand tetracycline. The antibiotics may be used to exert a selectionpressure to select for particular bacteria which contain an antibioticresistance gene and/or to select for Gram positive bacteria (e.g.,Streptococci). This can therefore be used to maintain selection pressurefor the bacteria expressing the desired cps. For example, the antibioticaztrianam is effective against Gram negative, but not Gram positivebacteria. Antifoaming agents are known in the art and may includemineral oil, medical oil, highly formulated polysiloxane glycolcopolymers, silicone compounds and emulsions, oxalkylated compounds,mineral oil/synthetic blends, glycol/ester blends, etc.

The culture may also include the addition of various other factors thatenhance growth, such as, lipids (such as long chain fatty acids such aslinoleic or oleic acid), steroids (such as cholesterol), purines andpyrimidines, vitamins and growth factors, amino acids (L- and/or D-form)and/or chemical elements or inorganic ions (such as Fe, K, Mg, Mn, Ca,Co, Cu, P and/or Zn).

If the growth medium contains additives obtained from animals, such asbovine serum albumin, these should be obtained from sources free oftransmissible spongiform encephalopathies to avoid contamination of themedium and eventually the cps.

Carbon Source

The type of carbon source used is not essential. Preferably a primarycarbon source is selected from the group consisting of glucose,fructose, lactose, sucrose, maltodextrins, starch, inulin, glycerol,vegetable oils such as soybean oil, hydrocarbons, alcohols such asmethanol and ethanol, organic acids such as acetate. More preferably thecarbon source is selected from glucose, glycerol, lactose, fructose,sucrose and soybean oil. The term “glucose” includes glucose syrups,i.e. glucose compositions comprising glucose oligomers. The carbonsource may be added to the culture as a solid or liquid. Preferably thecarbon source is controlled to avoid osmotic stress on the cells whichcan result in overfeeding. This is usually achieved by not adding theentire carbon source required for the duration of the fermentation tothe initial batch culture. The carbon source is also controlled to avoiddepletion which can result in growth limitation and pigment production(Ref. 13).

Nitrogen Source

The type of nitrogen source used is not essential. Preferably, thenitrogen source is selected from urea, ammonium hydroxide, ammoniumsalts (such as ammonium sulphate, ammonium phosphate, ammonium chlorideand ammonium nitrate), other nitrates, amino acids such as glutamate andlysine, yeast extract, yeast autolysates, yeast nitrogen base, proteinhydrolysates (including, but not limited to peptones, caseinhydrolysates such as tryptone and casamino acids), soybean meal, Hy-Soy,tryptic soy broth, cotton seed meal, malt extract, corn steep liquor andmolasses. More preferably, a nitrogen source is selected from ammoniumhydroxide, ammonium sulphate, ammonium chloride and ammonium phosphate.Most preferably, the nitrogen source is ammonium hydroxide. The use ofammonium hydroxide as a nitrogen source has the advantage that ammoniumhydroxide additionally can function as a pH-controlling agent.

If ammonium sulphate and/or ammonium phosphate are used as a nitrogensource, at least a portion of the sulfur and/or phosphorus requirementof the microorganism may be met.

Phosphorus Source

As noted above, phosphorus may be added to the growth medium. Thephosphorus may be in the form of a salt, in particular it may be addedas a phosphate (such as ammonium phosphate as noted above) orpolyphosphate. If a polyphosphate is used, it may be in the form of aphosphate glass, such as sodium polyphosphate (Ref. 14). Such phosphateglasses are useful as their solubility properties are such thatconcentrated nutrient media can be prepared with no resultingprecipitation upon mixing.

Other Variables

The temperature of the culture is kept between 30-45° C. (e.g., at 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44° C.). Preferably thetemperature is about 36° C. Thus, it may be necessary to heat or coolthe vessel containing the culture to ensure a constant culturetemperature is maintained. The temperature may be used to control thedoubling time (t_(d)), thus for a given culture process, the temperaturemay be different at different phases (i.e. the batch phase, fed batchphase and carbon feed phase).

The oxygen feed of the culture may be controlled. Oxygen may be suppliedas air, enriched oxygen, pure oxygen or any combination thereof. Methodsof monitoring oxygen concentration are known in the art. Oxygen may bedelivered at a certain feed rate or may be delivered on demand bymeasuring the dissolved oxygen content of the culture and feedingaccordingly with the intention of maintaining a constant dissolvedoxygen content.

The rate of agitation or aeration may also be controlled. This ensuresthat nutrients and oxygen are transferred around the bioreactor in whichthe culture is contained. The relative velocity between the nutrientsolution and the individual cell should be around 0.5m/sec (e.g., 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 s).

As noted above, the pH of the culture may be controlled by the additionof acid or alkali. As pH will typically drop during culture, preferablyalkali is added. Examples of suitable alkalis include NaOH and NH₄OH.

All of these variables may be controlled by the computer, computer-aideddevice or control algorithm as mentioned above. The alteration of thesevariables may be used to control the doubling time of the culture.

Polysaccharide Preparation

Methods for preparing capsular saccharides from bacteria are well knownin the art, e.g., see references 15, 16, 17, etc. For GBS, the followingmethods may be used (see also Ref. 18). In particular, the methods ofthe invention for purifying a capsular polysaccharide may be used. Asdiscussed above, these methods of the invention may include one or moreof the following steps.

Starting Material

Generally, a small amount of capsular polysaccharide is released intothe culture medium during bacterial growth, and so the starting materialmay thus be the supernatant from a centrifuged bacterial culture. Moretypically, however, the starting material will be prepared by treatingthe capsulated bacteria themselves (or material containing the bacterialpeptidoglycan), such that the capsular saccharide is released. Cps canbe released from bacteria by various methods, including chemical,physical or enzymatic treatment. Thus, an aqueous preparation ofpolysaccharide can be treated prior to the initial protein/nucleic acidprecipitation reaction.

A typical chemical treatment is base extraction (Ref. 19) (e.g., usingsodium hydroxide), which can cleave the phosphodiester linkage betweenthe capsular saccharide and the peptidoglycan backbone. As basetreatment de-N-acetylates the capsular saccharide, however, laterre-N-acetylation may be necessary.

A typical enzymatic treatment involves the use of both mutanolysin and13-N-acetylglucosaminidase (Ref. 20). These act on the bacterialpeptidoglycan to release the capsular saccharide for use with theinvention, but also lead to release of the group-specific carbohydrateantigen. An alternative enzymatic treatment involves treatment with atype II phosphodiesterase (PDE2). PDE2 enzymes can cleave the samephosphates as sodium hydroxide (see above) and can release the capsularsaccharide without cleaving the group-specific carbohydrate antigen andwithout de-N-acetylating the capsular saccharide, thereby simplifyingdownstream steps. PDE2 enzymes are therefore a preferred option forpreparing capsular saccharides.

A preferred starting material for the process of the invention isde-N-acetylated capsular polysaccharide, which can be obtained by baseextraction as described in U.S. Pat. No. 6,248,570 (Ref. 19). Anotherpreferred starting material is the product of PDE2 treatment ofStreptococcus. Such materials can be subjected to concentration (e.g.,ultrafiltration) prior to precipitation as mentioned below.

The starting material may be subjected to alcoholic precipitation ofcontaminating proteins and/or nucleic acids, as described below.

Alcoholic Precipitation and Cation Exchange

The Streptococcus capsular saccharide obtained after culture willgenerally be impure and will be contaminated with bacterial nucleicacids and proteins. These contaminants can be removed by sequentialovernight treatments with RNAse, DNAse and protease. However, as apreferred alternative, rather than remove these contaminantsenzymatically, alcoholic precipitation can be used. If necessary (e.g.,after base extraction), materials will usually be neutralized prior tothe precipitation.

The alcohol used to precipitate contaminating nucleic acids and/orproteins is preferably a lower alcohol, such as methanol, ethanol,propan-1-ol, propan-2-ol, butan-1-ol, butan-2-ol, 2-methyl-propan-1-ol,2-methyl-propan-2-ol, diols, etc. The selection of an appropriatealcohol can be tested empirically, without undue burden, but alcoholssuch as ethanol and isopropanol (propan-2-ol) are preferred, rather thanalcohols such as phenol.

The alcohol is preferably added to the polysaccharide suspension to givea final alcohol concentration of between 10% and 50% (e.g., around 30%).The most useful concentrations are those which achieve adequateprecipitation of contaminants without also precipitating thepolysaccharide. The optimum final alcohol concentration may depend onthe bacterial serotype from which the polysaccharide is obtained, andcan be determined by routine experiments without undue burden.Precipitation of polysaccharides as ethanol concentrations >5% has beenobserved.

The alcohol may be added in pure form or may be added in a form dilutedwith a miscible solvent (e.g., water). Preferred solvent mixtures areethanol:water mixtures, with a preferred ratio of between around 70:30and around 95:5 (e.g., 75:25, 80:20, 85:15, 90:10).

The saccharide may also be treated with an aqueous metal cation.Monovalent and divalent metal cations are preferred, and divalentcations are particularly preferred, such as Mg, Mn, Ca, etc., as theyare more efficient at complex formation. Calcium ions are particularlyuseful, and so the alcohol mixture preferably includes soluble calciumion. These may be added to a saccharide/alcohol mixture in the form ofcalcium salts, either added as a solid or in an aqueous form. Thecalcium ions are preferably provided by the use of calcium chloride.

The calcium ions are preferably present at a final concentration ofbetween 10 and 500 mM (e.g., about 0.1 M). The optimum final Caconcentration may depend on the Streptococcus strain and serotype fromwhich the polysaccharide is obtained, and can be determined by routineexperiments without undue burden.

After alcoholic precipitation of contaminating proteins and/or nucleicacids, the capsular polysaccharide is left in solution. The precipitatedmaterial can be separated from the polysaccharide by any suitable means,such as by centrifugation. The supernatant can be subjected tomicrofiltration, and in particular to dead-end filtration (perpendicularfiltration) in order to remove particles that may clog filters in latersteps (e.g., precipitated particles with a diameter greater than 0.22μm). As an alternative to dead-end filtration, tangentialmicrofiltration can be used. For example, tangential microfiltrationusing a 0.2 μm cellulose membrane may be used. The step of tangentialmicrofiltration is typically followed by filtration using a 0.45/0.2 μmfilter.

Diailtration

A step of diafiltration may be used. For example, if the method includesthe alcoholic precipitation and cation exchange described above, thenthis step may be carried out after the precipitation of proteins and/ornucleic acids. Similarly, if the method includes the step of cationicdetergent treatment described below, then this diafiltration step may becarried out before the detergent-mediated precipitation. In the methodsof the invention that include filtration using an adherent filter, e.g.filtration with a protein adherent filter, this diafiltration step maybe carried out before that filtration. Typically, a step ofdiafiltration is used after the precipitation of proteins and/or nucleicacids, and before the detergent-mediated precipitation or filtrationusing an adherent filter, e.g. a protein adherent filter.

The diafiltration step is particularly advantageous if base extractionor phosphodiesterase was used for release of the capsular saccharide, asthe group specific saccharide will also have been hydrolyzed, to givefragments much smaller than the intact capsular saccharide. These smallfragments can be removed by the diafiltration step.

Tangential flow diafiltration is typical. The filtration membrane shouldthus be one that allows passage of hydrolysis products of thegroup-specific antigen while retaining the capsular polysaccharide. Acut-off in the range 10 kDa-30 kDa is typical. Smaller cut-off sizes canbe used, as the hydrolysis fragments of the group-specific antigen aregenerally around 1 kDa (5-mer, 8-mer and 11-mer saccharides), but thehigher cut-off advantageously allows removal of other contaminantswithout leading to loss of the capsular saccharide.

At least 5 cycles of tangential flow diafiltration are usuallyperformed, e.g., 6, 7, 8, 9, 10, 11 or more. Typically. 2 cycles oftangential flow diafiltration are performed. Between the first andsecond cycles, the retentate of the first diafiltration cycle may betreated with an acetic acid/sodium acetate solution. The resultantsuspension may be filtered to remove precipitate, e.g. using a 0.45 μmfilter. The suspension may also, or in addition, be filtered using a 0.2μm filter.

The diafiltration may be followed by further filtration using a 0.45/0.2μm filter.

Cationic Detergent Treatment

Many techniques for precipitating soluble polysaccharides are known inthe art. The saccharide may optionally be precipitated using one or morecationic detergents, though preferred embodiments of the purificationwill exclude detergent precipitation. Treating a mixture of the capsularsaccharide and group-specific saccharide with a cationic detergent leadsto preferential precipitation of the capsular saccharide, therebyadvantageously and conveniently minimizing contamination by thegroup-specific saccharide.

Particularly preferred detergents for use in the process of theinvention are tetrabutylammonia and cetyltrimethylammonia salts (e.g.,the bromide salts). Cetyltrimethylammonia bromide (CTAB) is particularlypreferred (Ref. 21). CTAB is also known as hexadecyltrimethylammoniabromide, cetrimonium bromide, Cetavlon and Centimide. Other detergentsinclude hexadimethrine bromide and myristyltrimethylammonia salts.

The detergent-mediated precipitation step is preferably selective forthe capsular polysaccharide.

Advantageously, the optional detergent precipitation may use a detergentsuch as CTAB that interacts with sialic acid residues in the saccharide,e.g., via carboxyl groups in the sialic acid. The detergent will thuspreferentially precipitate the sialic acid-containing capsularsaccharides, and particularly longer saccharides within a mixedpopulation, thus minimizing contamination by saccharides whoseantigenically-important sialic acids may have been damaged in earliertreatment steps.

Re-Solubilization

When an optional detergent precipitation step is used, thepolysaccharide (typically in the form of a complex with the cationicdetergent) can be re-solubilized, either in aqueous medium or inalcoholic medium. For aqueous re-solubilization, the CTA-cation in theprecipitate will generally be replaced by a metal cation; for alcoholicre-solubilization, the CTA-cation will generally be retained. The choiceof aqueous or alcoholic re-solubilization may depend on the GBS serotypefrom which the polysaccharide is obtained, and on any contaminants stillpresent at this stage. For example, pigments are sometimes present inthe precipitated pellet, and these can effectively be removed byalcoholic re-solubilization followed by carbon filtration.

A typical aqueous medium for re-solubilization will include a metalcation. Monovalent and divalent metal cations are preferred, anddivalent cations are particularly preferred, such as Mn, Ca, etc.Calcium ions are particularly useful, and so re-solubilizationpreferably uses Ca, provided by the use of calcium chloride. A Caconcentration of between 10 and 500 mM (e.g., about 0.1 M) is preferred.The optimum final Ca concentration may depend on the Streptococcusserotype from which the polysaccharide is obtained, and can bedetermined by routine experiments without undue burden.

A typical alcoholic medium for re-solubilization is based on ethanol.The same alcohols used for precipitation of nucleic acids and/orproteins can be used, but the concentration required for precipitationof the capsular saccharide will generally be higher, e.g., the alcoholis preferably added to give a final alcohol concentration of between 70%and 95% (e.g. around 70%, 75%, 80%, 85%, 90% or 95%). The optimum finalalcohol concentration may depend on the Streptococcus serotype fromwhich the polysaccharide is obtained. To achieve the high alcoholconcentrations then it is preferred to add alcohol with a low watercontent, e.g., 96% ethanol.

Re-solubilization will typically occur at room temperature. Acidicconditions are preferably avoided, and re-solubilization will typicallytake place at about pH 7.

The re-solubilized material is highly purified relative to thepre-precipitation suspension.

One preferred method for preparing the saccharides involvespolysaccharide precipitation followed by solubilization of theprecipitated polysaccharide using a lower alcohol as described above.After re-solubilization, the polysaccharide may be further treated toremove contaminants.

This is particularly important in situations where even minorcontamination is not acceptable (e.g., for human vaccine production).This will typically involve one or more steps of filtration, e.g., depthfiltration, filtration through activated carbon may be used, sizefiltration and/or ultrafiltration. Once filtered to remove contaminants,the polysaccharide may be precipitated for further treatment and/orprocessing. This can be conveniently achieved by exchanging cations(e.g., by the addition of calcium or sodium salts).

Filtration with an Adherent Filter

In preferred embodiments, the purification of the capsularpolysaccharides will further include a step whereby protein and/or DNAcontaminants are removed by filtration with a filter, e.g. a proteinadherent filter, to which protein and/or DNA adheres, but to which thecapsular polysaccharide does not adhere or only weakly adheres. Apreferred example of such filter is a carbon filter. Suitable adherentfilters are described above.

The filtration using an adherent filter may be followed by furtherfiltration using a 0.45/0.2 μm filter.

Re-N-Acetylation

A step of re-N-acetylation may be carried out, for example after a stepof filtration using an adherent filter or, if present, furtherfiltration step. Re-N-acetylation may be advantageous if sialic acidresidues in the GBS capsular saccharides have been de-N-acetylated, forexample during the base treatment described above. Controlledre-N-acetylation can conveniently be performed using a reagent such asacetic anhydride (CH₃CO)₂O, e.g. in 5% ammonium bicarbonate [Wessels etal. (1989) Inject Immun 57:1089-94].

Further Diafiltration

A further step of diafiltration may be carried out, for example afterre-N-acetylation. The diafiltration may be carried out as describedabove in the section entitled “Diafiltration”.

The diafiltration may be followed by further filtration using a 0.45/0.2μm filter.

Final Material

The polysaccharide is preferably finally prepared as a dried powder,ready for conjugation.

Conjugate Preparation

After culture of bacteria and preparation of capsular polysaccharides,the saccharide are conjugated to carrier protein(s). In general,covalent conjugation of saccharides to carriers enhances theimmunogenicity of saccharides as it converts them from T-independentantigens to T-dependent antigens, thus allowing priming forimmunological memory. Conjugation is particularly useful for pediatricvaccines (e.g., ref. 22) and is a well known technique (e.g., reviewedin refs. 23 to 31)

Preferred carrier proteins are bacterial toxins or toxoids, such asdiphtheria toxoid or tetanus toxoid. The CRM1 97 mutant of diphtheriatoxin (Refs 32-34) is a particularly preferred carrier for, as is adiphtheria toxoid. Other suitable carrier proteins include the Nmeningitidis outer membrane protein (Ref. 35), synthetic peptides (Refs.36,37), heat shock proteins (Refs. 3 8,39), pertussis proteins (Refs.40,41), cytokines (Ref. 42), lymphokines (Ref. 42), hormones (Ref. 42),growth factors (Ref. 42), artificial proteins comprising multiple humanCD4 T cell epitopes from various pathogen-derived antigens (Ref. 43)such as N19 (Ref. 44), protein D from H. influenzae (Ref. 45,46),pneumococcal surface protein PspA (Ref. 47), pneumolysin (Ref. 48),iron-uptake proteins (Ref. 49), toxin A or B from C. difficile (Ref 50),a GBS protein (see below) (Ref. 51), etc. Attachment to the carrier ispreferably via a —NH2 group. e.g., in the side chain of a lysine residuein a carrier protein, or of an arginine residue. Where a saccharide hasa free aldehyde group then this can react with an amine in the carrierto form a conjugate by reductive amination. Such a conjugate may becreated using reductive amination involving an oxidized galactose in thesaccharide (from which an aldehyde is formed) and an amine in thecarrier or in the linker. Attachment may also be via a —SH group, e.g.,in the side chain of a cysteine residue.

It is possible to use more than one carrier protein, e.g., to reduce therisk of carrier suppression. Thus different carrier proteins can be usedfor different Streptococcus strains or serotypes, e.g., GBS serotype Iasaccharides might be conjugated to CRM197 while serotype Ib saccharidesmight be conjugated to tetanus toxoid. It is also possible to use morethan one carrier protein for a particular saccharide antigen, e.g.,serotype III saccharides might be in two groups, with some conjugated toCRM197 and others conjugated to tetanus toxoid. In general, however, itis preferred to use the same carrier protein for all saccharides.

A single carrier protein might carry more than one saccharide antigen(Refs. 52, 53). For example, a single carrier protein might haveconjugated to it saccharides from serotypes Ia and Ib. To achieve thisgoal, different saccharides can be mixed prior to the conjugationreaction. In general, however, it is preferred to have separateconjugates for each serogroup, with the different saccharides beingmixed after conjugation The separate conjugates may be based on the samecarrier.

Conjugates with a saccharide:protein ratio (w/w) of between excessprotein (e.g., 1:5) and excess saccharide (e.g., 5:1) are preferred.Ratios between 1:2 and 5:1 are preferred, as are ratios between 1:1.25and 1:2.5. Ratios between 1:1 and 4:1 are also preferred. With longersaccharide chains, a weight excess of saccharide is typical. In general,the invention provides a conjugate, wherein the conjugate comprises aStreptococcus, preferably a S agalactiae capsular saccharide moietyjoined to a carrier, wherein the weight ratio of saccharide: carrier isat least 2:1.

Compositions may include a small amount of free carrier. When a givencarrier protein is present in both free and conjugated form in acomposition of the invention, the unconjugated form is preferably nomore than 5% of the total amount of the carrier protein in thecomposition as a whole, and more preferably present at less than 2% byweight.

Any suitable conjugation reaction can be used, with any suitable linkerwhere necessary.

The saccharide will typically be activated or functionalized prior toconjugation. Activation may involve, for example, cyanylating reagentssuch as CDAP (e.g., 1.-cyano-4-dimethylamino pyridiniumtetrafluoroborate (Refs. 54, 55, etc.)). Other suitable techniques usecarbodiimides, hydrazides, active esters, norborane, p-nitrobenzoicacid, N-hydroxysuccinimide, S-NHS, EDC, and TSTU (see also theintroduction to reference 29).

Linkages via a linker group may be made using any known procedure, forexample, the procedures described in references 56 and 57. One type oflinkage involves reductive amination of the polysaccharide, coupling theresulting amino group with one end of an adipic acid linker group, andthen coupling a protein to the other end of the adipic acid linker group(Refs. 27, 58, 59). Other linkers include B-propionamido (Ref. 60),nitrophenyl-ethylamine (Ref 61), haloacyl halides (Ref 62), glycosidiclinkages (Ref. 63), 6-aminocaproic acid (Ref 64), ADH (Ref. 65), C4 toC12 moieties (Ref. 66), etc. As an alternative to using a linker, directlinkage can be used. Direct linkages to the protein may compriseoxidation of the polysaccharide followed by reductive amination with theprotein, as described in, for example, references 67 and 68.

A process involving the introduction of amino groups into the saccharide(e.g., by replacing terminal ═O groups with —NH2) followed byderivatization with an adipic diester (e.g., adipic acidN-hydroxysuccinimido diester) and reaction with carrier protein ispreferred. Another preferred reaction uses CDAP activation with aprotein D carrier.

After conjugation, free and conjugated saccharides can be separated.There are many suitable methods, including hydrophobic chromatography,tangential ultrafiltration, diafiltration, etc. (see also refs. 69 & 70,etc.).

Where the composition of the invention includes a depolymerizedoligosaccharide, it is preferred that depolymerization precedesconjugation, e.g., is before activation of the saccharide.

In one preferred conjugation method, a saccharide is reacted with adipicacid dihydrazide. For serogroup A, carbodiimide may also be added atthis stage. After a reaction period, sodium cyanoborohydride is added.Derivatized saccharide can then be prepared, e.g., by ultrafiltration.

The derivatized saccharide is then mixed with carrier protein (e.g.,with a diphtheria toxoid), and carbodiimide is added. After a reactionperiod, the conjugate can be recovered.

Other Steps

As well as including the steps described above, methods of the inventionmay include further steps. For example, the methods may include a stepof depolymerization of the capsular saccharides, after they are preparedfrom the bacteria but before conjugation. Depolymerization reduces thechain length of the saccharides and may not be good for GBS. ForStreptococcus, especially GBS, longer saccharides tend to be moreimmunogenic than shorter ones (Ref. 71).

After conjugation, the level of unconjugated carrier protein may bemeasured. One way of making this measurement involves capillaryelectrophoresis (Ref. 72) (e.g., in free solution), or micellarelectrokinetic chromatography (Ref. 73).

After conjugation, the level of unconjugated saccharide may be measured.One way of making this measurement involves HPAEC-PAD (Ref. 69).

After conjugation, a step of separating conjugated saccharide fromunconjugated saccharide may be used. One way of separating thesesaccharides is to use a method that selectively precipitates onecomponent. Selective precipitation of conjugated saccharide ispreferred, to leave unconjugated saccharide in solution, e.g., by adeoxycholate treatment (Ref. 69).

After conjugation, a step of measuring the molecular size and/or molarmass of a conjugate may be carried out. In particular, distributions maybe measured. One way of making these measurements involves sizeexclusion chromatography with detection by multiangle light scatteringphotometry and differential refractometry (SEC-MALS/RI) (Ref. 74).

Coniugate Combinations

Individual conjugates can be prepared as described above, for anyPneumococcus serogroup.

Preferably conjugates are prepared for one or more of serogroups 1, 3,4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F. The individual conjugates canthen be mixed, in order to provide a polyvalent mixture.

It is also possible to mix a selected number of conjugates to provide abivalent, trivalent, tetravalent, 5-valent, 6-valent, 7-valent or11-valent mixture (e.g., to mix 1+3+4+5+6B+7F+9V+14+18C+19F+23F,4+6B+9V+14+18C+19F+23F or 1+4+6B+9V+14+1 8C+19F+23F, etc.).

For GBS, conjugates are preferably prepared from one or more ofserogroups Ia, Ib or III.

Conjugates may be mixed by adding them individually to a bufferedsolution. A preferred solution is phosphate buffered physiologicalsaline (final concentration 10 mM sodium phosphate). A preferredconcentration of each conjugate (measured as saccharide) in the finalmixture is between 1 and 20 μg/ml e.g., between 5 and 15 μg/ml, such asaround 8 μg/ml. An optional aluminum salt adjuvant may be added at thisstage (e.g., to give a final Al³⁺ concentration of between 0.4 and 0.5mg/ml).

After mixing, the mixed conjugates can be sterile filtered.

Pharmaceutical Compositions

Conjugates prepared by methods of the invention can be combined withpharmaceutically acceptable carriers. Such carriers include any carrierthat does not itself induce the production of antibodies harmful to theindividual receiving the composition. Suitable carriers are typicallylarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, sucrose, trehalose, lactose, and lipidaggregates (such as oil droplets or liposomes). Such carriers are wellknown to those of ordinary skill in the art. The vaccines may alsocontain diluents, such as water, saline, glycerol, etc. Additionally,auxiliary substances, such as wetting or emulsifying agents, pHbuffering substances, and the like, may be present. Sterilepyrogen-free, phosphate-buffered physiologic saline is a typicalcarrier. A thorough discussion of pharmaceutically acceptable excipientsis available in reference 75.

Compositions may include an antimicrobial, particularly if packaged in amultiple dose format.

Compositions may comprise detergent, e.g., a Tween (polysorbate), suchas TWEEN 80™. Detergents are generally present at low levels, (e.g.,>0.01%).

Compositions may include sodium salts (e.g., sodium chloride) to givetonicity. A concentration of 10±2 mg/ml NaCl is typical.

Compositions will generally include a buffer. A phosphate buffer istypical.

Compositions may comprise a sugar alcohol (e.g., mannitol) or adisaccharide (e.g., sucrose or trehalose) e.g., at around 15-30 mg/ml(e.g., 25 mg/ml), particularly if they are to be lyophilized or if theyinclude material which has been reconstituted from lyophilized material.The pH of a composition for lyophilization may be adjusted to around 6.1prior to lyophilization.

Conjugates may be administered in conjunction with otherimmunoregulatory agents. In particular, compositions will usuallyinclude a vaccine adjuvant. Adjuvants which may be used in compositionsof the invention include, but are not limited to:

A. Mineral-Containing Compositions

Mineral containing compositions suitable for use as adjuvants in theinvention include mineral salts, such as aluminum salts and calciumsalts (or mixtures thereof). The invention includes mineral salts suchas hydroxides (e.g., oxyhydroxides), phosphates (e.g.,hydroxyphosphates, orthophosphates), sulphates, etc. (Ref e.g., seechapters 8 & 9 of ref. 76), or mixtures of different mineral compounds,with the compounds taking any suitable form (e.g., gel, crystalline,amorphous, etc.), and with adsorption being preferred. The mineralcontaining compositions may also be formulated as a particle of metalsalt (Ref. 77).

Aluminum phosphates are particularly preferred, particularly incompositions which include a H. influenzae saccharide antigen, and atypical adjuvant is amorphous aluminum hydroxyphosphate with PO₄/Almolar ratio between 0.84 and 0.92, included at 0.6 mg Al⁺³/ml.

Adsorption with a low dose of aluminum phosphate may be used e.g.,between 50 and 100 μg Al³⁺ per conjugate per dose. Where there is morethan one conjugate in a composition, not all conjugates need to beadsorbed.

Calcium salts include calcium phosphate (e.g. the “CAP” particlesdisclosed in ref. 258. Aluminum salts include hydroxides, phosphates,sulfates, etc., with the salts taking any suitable form (e.g. gel,crystalline, amorphous, etc.). Adsorption to these salts is preferred.The mineral containing compositions may also be formulated as a particleof metal salt [77]. Aluminum salt adjuvants are described in more detailbelow.

The adjuvants known as aluminum hydroxide and aluminum phosphate may beused. These names are conventional, but are used for convenience only,as neither is a precise description of the actual chemical compoundwhich is present (e.g. see chapter 9 of reference 76). The invention canuse any of the “hydroxide” or “phosphate” adjuvants that are in generaluse as adjuvants.

The adjuvants known as “aluminium hydroxide” are typically aluminiumoxyhydroxide salts, which are usually at least partially crystalline.Aluminium oxyhydroxide, which can be represented by the formula AlO(OH),can be distinguished from other aluminium compounds, such as aluminiumhydroxide Al(OH)₃, by infrared (IR) spectroscopy, in particular by thepresence of an adsorption band at 1070 cm⁻¹ and a strong shoulder at3090-3100 cm⁻¹ [chapter 9 of ref 76]. The degree of crystallinity of analuminium hydroxide adjuvant is reflected by the width of thediffraction band at half height (WHH), with poorly crystalline particlesshowing greater line broadening due to smaller crystallite sizes. Thesurface area increases as WHH increases, and adjuvants with higher WHHvalues have been seen to have greater capacity for antigen adsorption. Afibrous morphology (e.g. as seen in transmission electron micrographs)is typical for aluminium hydroxide adjuvants. The pI of aluminiumhydroxide adjuvants is typically about 11 i.e. the adjuvant itself has apositive surface charge at physiological pH. Adsorptive capacities ofbetween 1.8-2.6 mg protein per mg Al⁺⁺⁺ at pH 7.4 have been reported foraluminium hydroxide adjuvants.

The adjuvants known as “aluminium phosphate” are typically aluminiumhydroxyphosphates, often also containing a small amount of sulfate (i.e.aluminium hydroxyphosphate sulfate). They may be obtained byprecipitation, and the reaction conditions and concentrations duringprecipitation influence the degree of substitution of phosphate forhydroxyl in the salt. Hydroxyphosphates generally have a PO₄/AI molarratio between 0.3 and 1.2. Hydroxyphosphates can be distinguished fromstrict AlPO₄ by the presence of hydroxyl groups. For example, an IRspectrum band at 3164 cm⁻¹ (e.g. when heated to 200° C.) indicates thepresence of structural hydroxyls [ch. 9 of ref. 76].

The PO₄/Al³⁺ molar ratio of an aluminium phosphate adjuvant willgenerally be between 0.3 and 1.2, preferably between 0.8 and 1.2, andmore preferably 0.95+0.1. The aluminium phosphate will generally beamorphous, particularly for hydroxyphosphate salts. A typical adjuvantis amorphous aluminium hydroxyphosphate with PO₄/AI molar ratio between0.84 and 0.92, included at 0.6 mg Al³⁺/ml. The aluminium phosphate willgenerally be particulate (e.g. plate like morphology as seen intransmission electron micrographs). Typical diameters of the particlesare in the range 0.5-20 μm (e.g. about 5-10 μm) after any antigenadsorption. Adsorptive capacities of between 0.7-1.5 mg protein per mgAl⁺⁺⁺ at pH 7.4 have been reported for aluminium phosphate adjuvants.

The point of zero charge (PZC) of aluminium phosphate is inverselyrelated to the degree of substitution of phosphate for hydroxyl, andthis degree of substitution can vary depending on reaction conditionsand concentration of reactants used for preparing the salt byprecipitation. PZC is also altered by changing the concentration of freephosphate ions in solution (more phosphate=more acidic PZC) or by addinga buffer such as a histidine buffer (makes PZC more basic). Aluminiumphosphates used according to the invention will generally have a PZC ofbetween 4.0 and 7.0, more preferably between 5.0 and 6.5 e.g. about 5.7.

Suspensions of aluminium salts used to prepare compositions of theinvention may contain a buffer (e.g. a phosphate or a histidine or aTris buffer), but this is not always necessary. The suspensions arepreferably sterile and pyrogen free. A suspension may include freeaqueous phosphate ions e.g. present at a concentration between 1.0 and20 mM, preferably between 5 and 15 mM, and more preferably about 10 mM.The suspensions may also comprise sodium chloride.

The invention can use a mixture of both an aluminium hydroxide and analuminium phosphate, as in DARONRIX™. In this case there may be morealuminium phosphate than hydroxide e.g. a weight ratio of at least 2:1e.g. >5:1, >6:1, >7:1, >8:1, >9:1, etc.

The concentration of Al⁺⁺⁺ in a composition for administration to apatient is preferably less than 10 mg/ml e.g. <5 mg/ml, <4 mg/ml, <3mg/ml, <2 mg/ml, <1 mg/ml, etc. A preferred range is between 0.3 and 1mg/ml. A maximum of 0.85 mg/dose is preferred.

B. Oil Emulsions

Oil emulsion compositions suitable for use as adjuvants in the inventioninclude squalene-water emulsions, such as MF59® (Ref. Chapter 10 of ref.76; see also ref. 78) (5% Squalene, 0.5% TWEEN*) 80, and 0.5% Span® 85,formulated into submicron particles using a microfluidizer). CompleteFreund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may alsobe used.

The oil droplets in the emulsion are generally less than 5 μm indiameter, and may even have a sub-micron diameter, with these smallsizes being achieved with a microfluidiser to provide stable emulsions.Droplets with a size less than 220 nm are preferred as they can besubjected to filter sterilization.

C. Saponin Formulations (Ref. Chapter 22 of Ref 76)

Saponin formulations may also be used as adjuvants in the invention.Saponins are a heterologous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, stems, roots and evenflowers of a wide range of plant species. Saponin from the bark of theQuilaia saponaria Molina tree have been widely studied as adjuvants.Saponin can also be commercially obtained from Smilax ornata(sarsaprilla), Gypsophilla paniculata (brides veil), and Saponariaofficianalis (soap root). Saponin adjuvant formulations include purifiedformulations, such as QS21, as well as lipid formulations, such asISCOMs. QS21 is marketed as STIMULON™.

Saponin compositions have been purified using HPLC and RP-HIPLC.Specific purified fractions using these techniques have been identified,including QS7, QS17, QS1 8. QS21, QH-A, QH-B and QH-C. Preferably, thesaponin is QS21. A method of production of QS21 is disclosed in ref. 79.Saponin formulations may also comprise a sterol, such as cholesterol(Ref. 80).

Combinations of saponins and cholesterols can be used to form uniqueparticles called immunostimulating complexes (ISCOMs) (Ref. chapter 23of ref. 76). ISCOMs typically also include a phospholipid such asphosphatidylethanolamine or phosphatidyiclioline. Any known saponin canbe used in ISCOMs. Preferably, the ISCOM includes one or more of QuilA,QHA & QHC. ISCOMs are further described in refs. 80-82. Optionally, theISCOMS may be devoid of additional detergent (Ref. 83).

A review of the development of saponin based adjuvants can be found inrefs. 84 & 85.

D. Virosomes and Virus-Like Particles

Virosomes and virus-like particles (VLPs) can also be used as adjuvantsin the invention. These structures generally contain one or moreproteins from a virus optionally combined or formulated with aphospholipid. They are generally non-pathogenic, non-replicating andgenerally do not contain any of the native viral genome. The viralproteins may be recombinantly produced or isolated from whole viruses.These viral proteins suitable for use in virosomes or VLPs includeproteins derived from influenza virus (such as HA or NA). Hepatitis Bvirus (such as core or capsid proteins), Hepatitis E virus, measlesvirus, Sindbis virus. Rotavirus, Foot-and-Mouth Disease virus,Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages,Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, andTy (such as retrotransposon Ty protein pl). VLPs are discussed furtherin refs. 86-91. Virosomes are discussed further in, for example, ref.92.

E. Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial ormicrobial derivatives such as non-toxic derivatives of enterobacteriallipopolysaccharide (LPS), Lipid A derivatives, immunostimulatoryoligonucleotides and ADP-ribosylating toxins and detoxified derivativesthereof.

Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylatedmonophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred“small particle” form of 3 De-O-acylated monophosphoryl lipid A isdisclosed in ref. 93. Such “small particles” of 3dMPL are small enoughto be sterile filtered through a 0.22 μm membrane (Ref. 93). Othernon-toxic LPS derivatives include monophosphoryl lipid A mimics, such asaminoalkyl glucosaminide phosphate derivatives e.g., RC-529 (Ref.94,95).

Lipid A derivatives include derivatives of lipid A from Escherichia colisuch as OM-174. OM-174 is described for example in refs. 96 & 97.

Immunostimulatory oligonucleotides suitable for use as adjuvants in theinvention include nucleotide sequences containing a CpG motif (adinucleotide sequence containing an unmethylated cytosine linked by aphosphate bond to a guanosine). Double-stranded RNAs andoligonucleotides containing palindromic or poly(dG) sequences have alsobeen shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such asphosphorothioate modifications and can be double-stranded orsingle-stranded. References 98, 99 and 100 disclose possible analogsubstitutions, e.g., replacement of guanosine with2′-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotidesis further discussed in refs. 101-106.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT orTTCGTT (Ref. 107). The CpG sequence may be specific for inducing a Th1immune response, such as a CpG-A ODN, or it may be more specific forinducing a B cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs arediscussed in refs. 108-110. Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end isaccessible for receptor recognition. Optionally, two CpG oligonucleotidesequences may be attached at their 3′ ends to form “immunomers.” See,for example, refs. 107 & 111-113.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof maybe used as adjuvants in the invention. Preferably, the protein isderived from E. coli (E. coli heat labile enterotoxin “LT”), cholera(“CT”), or pertussis (PT”). The use of detoxified ADP-ribosylatingtoxins as mucosal adjuvants is described in ref. 114 and as parenteraladjuvants in ref. 115. The toxin or toxoid is preferably in the form ofa holotoxin, comprising both A and B subunits. Preferably, the A subunitcontains a detoxifying mutation; preferably the B subunit is notmutated. Preferably, the adjuvant is a detoxified LT mutant such asLT-K63, LT-R72, and LT-G192. The use of ADP-ribosylating toxins anddetoxified derivatives thereof, particularly LT-K63 and LT-R72, asadjuvants can be found in refs. 116-123. Numerical reference for aminoacid substitutions is preferably based on the alignments of the A and Bsubunits of ADP-ribosylating toxins set forth in ref. 124, specificallyincorporated herein by reference in its entirety.

F. Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the inventioninclude cytokines, such as interleukins (e.g., IL-1, IL-2, 11-4, IL-5,IL-6, IL-7, IL-12 (Ref. 125), etc.) (Ref. 126), interferons (e.g.,interferon-T), macrophage colony stimulating factor, and tumor necrosisfactor. A preferred immunomodulator is IL-12.

G. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in theinvention. Suitable bioadhesives include esterified hyaluronic acidmicrospheres (Ref. 127) or mucoadhesive such as cross-linked derivativesof poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone,polysaccharides and carboxymethylcellulose. Chitosan and derivativesthereof may also be used as adjuvants in the invention (Ref. 128).

H. Microparticles

Microparticles may also be used as adjuvants in the invention.Microparticles (i.e., a particle of ˜100 nm to ˜150 Atm in diameter,more preferably ˜200 nm to ˜33 μm in diameter, and most preferably ˜500nm to ˜10 μm in diameter) formed from materials that are biodegradableand non-toxic (e.g., a poly(α-hydroxy acid), a polyhydroxybutyric acid,a polyorthoester, a polyarthydride, a polycaprolactone, etc.), withpoly(lactide-co-glycolide) are preferred, optionally treated to have anegatively charged surface (e.g., with SDS) or a positively-chargedsurface (e.g., with a cationic detergent, such as CTAB).

I. Liposomes (Chapters 13 & 14 of Ref 76)

Examples of liposome formulations suitable for use as adjuvants aredescribed in refs. 129-131.

J. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations

Adjuvants suitable for use in the invention include polyoxyethyleneethers and polyoxyethylene esters (Ref. 132). Such formulations furtherinclude polyoxyethylene sorbitan ester surfactants in combination withan octoxynol (Ref. 133) as well as polyoxyethylene alkyl ethers or estersurfactants in combination with at least one additional non-ionicsurfactant such as an octoxynol (Ref. 134). Preferred polyoxyethyleneethers are selected from the following group: polyoxyethylene-9-laurylether (laureth 9), polyoxyethylene-9-steoryl ether,polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether,polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-laurvyl ether.

K Polyphosphazene (PCPP)

PCPP formulations are described, for example, in refs. 135 and 136.

L. Muramyl peptides

Examples of muramyl peptides suitable for use as adjuvants in theinvention include N-acetylmuramy-L-threonyl-D-isoglutamine (thr-MDP),N-acetylmuramyl-L-alanyl-D-isoglutamine (nor-MDP), andN-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3hydroxyphosphoryloxy)-ethylamine MTP-PE).

M. Imidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use adjuvants in theinvention include IMIQUAMOD™ and its homologues (e.g., RESIQUIMOD 3M™,described further in refs. 137 and 138.

The invention may also comprise combinations of aspects of one or moreof the adjuvants identified above. For example, the following adjuvantcompositions may be used in the invention: (1) a saponin and anoil-in-water emulsion (Ref. 139); (2) a saponin (e.g., QS21)+a non-toxicLPS derivative (e.g., 3dMPL) (Ref. 140): (3) a saponin (e.g., QS21)+anon-toxic LPS derivative (e.g., 3dMPL)+a cholesterol; (4) a saponin(e.g., QS2I)+3dMPL+IL-12 (optionally+a sterol) (Ref 141); (5)combinations of 3dMPL with, for example, QS21 and/or oil-in-wateremulsions (Ref. 142): (6) SAF, containing 10% squalane, 0.4% TWEEN 80™,5% pluronic-block polymer L121, and thr-MDP either microfluidized into asubmicron emulsion or vortexed to generate a larger particle sizeemulsion. (7) RIBI™ adjuvant system (RAS), (Ribi Immunochem) containing2% squalene, 0.2 TWEEN 80™, and one more bacterial cell wall componentsfrom the group consisting of monophosphorylipid A (MPL), trehalosedimcolate (TDM), and cell wall skeleton (CWS); preferably MPL+CWS(DETOX™); and (8) one or more mineral salts (such as an aluminum salt)+anon-toxic derivative of LPS (such as 3dMPL).

Other substances that act as immunostimulating agents are disclosed inchapter 7 of ref. 76.

The use of an aluminum hydroxide and/or aluminum phosphate adjuvant isparticularly preferred, and antigens are generally adsorbed to thesesalts. Calcium phosphate is another preferred adjuvant.

The composition may be sterile and/or pyrogen-free. Compositions may beisotonic with respect to humans.

Compositions may be presented in vials, or they may be presented inready-filled syringes. The syringes may be supplied with or withoutneedles. A syringe will include a single dose of the composition,whereas a vial may include a single dose or multiple doses. Injectablecompositions will usually be liquid solutions or suspensions.Alternatively, they may be presented in solid form (e.g., freeze-dried)for solution or suspension in liquid vehicles prior to injection.

Compositions may be packaged in unit dose form or in multiple dose form.For multiple dose forms, vials are preferred to pre-filled syringes.Effective dosage volumes can be routinely established, but a typicalhuman dose of the composition for injection has a volume of 0.5 ml.

Where a composition is to be prepared extemporaneously prior to use(e.g., where a component is presented in lyophilized form) and ispresented as a kit, the kit may comprise two vials, or it may compriseone ready-filled syringe and one vial, with the contents of the syringebeing used to reactivate the contents of the vial prior to injection.

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of antigen(s), as well as any other components, asneeded. By immunologically effective amount, it is meant that theadministration of that amount to an individual, either in a single doseor as part of a series, is effective for treatment or prevention. Thisamount varies depending upon the health and physical condition of theindividual to be treated, age, the taxonomic group of individual to betreated (e.g., non-human primate, primate, etc.), the capacity of theindividuals immune system to synthesize antibodies, the degree ofprotection desired, the formulation of the vaccine, the treatingdoctor's assessment of the medical situation, and other relevantfactors. It is expected that the amount will fall in a relatively broadrange that can be determined through routine trials, and a typicalquantity of each streptococcal conjugate in between 1 μg and 20 μg perconjugate (measured as saccharide).

Thus the invention provides a method for preparing a pharmaceuticalcomposition, comprising the steps of: (a) preparing a conjugate asdescribed above; (b) mixing the conjugate with one or morepharmaceutically acceptable carriers.

The invention further provides a method for preparing a pharmaceuticalproduct, comprising the steps of: (a) preparing a conjugate as describedabove: (b) mixing the conjugate with one or more pharmaceuticallyacceptable carriers; and (c) packaging the conjugate/carrier mixtureinto a container, such as a vial or a syringe, to give a pharmaceuticalproduct. Insertion into a syringe may be performed in a factory or in asurgery.

The invention also provides a method for preparing a pharmaceuticalcomposition from a saccharide-protein conjugate, comprising the step ofadmixing the conjugate with a pharmaceutically acceptable carrier,wherein the conjugate has been prepared by a process conjugation methodas described above. The conjugation method and the admixing step can beperformed at very different times by different people in differentplaces (e.g., in different countries).

The invention also provides a method for packaging a saccharide-proteinconjugate into a pharmaceutical product, wherein the conjugate has beenprepared by a process conjugation method as described above. Theconjugation method and the packaging step can be performed at verydifferent times by different people in different places (e.g., indifferent countries).

Pharmaceutical Uses

The invention also provides a method of treating a patient, comprisingadministering the composition to the patient. The patient may either beat risk from the disease themselves or may be a pregnant woman (matemalimmunization). The patient is preferably a human. The human can be ofany age e.g., <2 years old, from 2-11 years old, from 11-55 yearsold. >55 years old, etc.

The invention also provides the composition for use in therapy. Theinvention also provides the use of the composition in the manufacture ofa medicament for the treatment of disease. Preferably the disease isinfluenza or pneumonia.

Compositions will generally be administered directly to a patient.Direct delivery may be accomplished by parenteral injection (e.g.,transcutaneously, subcutaneously, intraperitoneally, intravenously,intramuscularly, or to the interstitial space of a tissue), or byrectal, oral, vaginal, optical, transdermal, intranasal, ocular, aural,pulmonary or other mucosal administration. Intramuscular administration(e.g., to the thigh or the upper arm) is preferred. Injection may be viaa needle (e.g., a hypodermic needle), but needle-free injection mayalternatively be used. A typical intramuscular dose is 0.5 ml.

The invention may be used to elicit systemic and/or mucosal immunity.

Dosage treatment can be a single dose schedule or a multiple doseschedule.

Multiple doses may be used in a primary immunization schedule and/or ina booster immunization schedule. A primary dose schedule may be followedby a booster dose schedule. Suitable timing between priming doses (e.g.,between 4-16 weeks), and between priming and boosting, can be routinelydetermined.

Bacterial infections affect various areas of the body and socompositions may be prepared in various forms. For example, thecompositions may be prepared as injectables, either as liquid solutionsor suspensions. Solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection can also be prepared (e.g., alyophilized composition). The composition may be prepared for topicaladministration, e.g., as an ointment, cream or powder. The compositionmay be prepared for oral administration. e.g., as a tablet or capsule,or as a syrup (optionally flavored). The composition may be prepared forpulmonary administration, e.g., as an inhaler, using a fine powder or aspray. The composition may be prepared as a suppository or pessary.

The composition may be prepared for nasal, aural or ocularadministration, e.g., as spray, drops, gel or powder (e.g., refs 143 &144). Injectable compositions are preferred.

Further antigenic components of compositions of the invention

The methods of the invention may also comprise the steps of mixing astreptococcal conjugate with one or more of the following otherantigens:

-   -   a saccharide antigen from Haemophilus influenzae B (e.g.,        chapter 14 of ref. 145).    -   a purified protein antigen from serogroup B of Neisseria        meningitidis.    -   an outer membrane preparation from serogroup B of Neisseria        meningitidis.    -   an antigen from hepatitis A virus, such as inactivated virus        (e.g., 46, 147).    -   an antigen from hepatitis B virus, such as the surface and/or        core, antigens (e.g., 147, 148).    -   a diphtheria antigen, such as a diphtheria toxoid (e.g., chapter        13 of ref. 145)    -   a tetanus antigen, such as a tetanus toxoid (e.g., chapter 27 of        ref 145).    -   an antigen from Bordetella pertussis, such as pertussis        holotoxin (PT) and filamentous hemagglutinin (FHA) from B.        pertussis, optionally also in combination with pertactin and/or        agglutinogens 2 and 3 (e.g., refs. 149 & 150; chapter 21 of ref.        145).    -   polio antigen(s) (e.g., 151, 152) such as IPV (chapter 24 of        ref. 145).    -   measles, mumps and/or rubella antigens (e.g., chapters 19, 20 &        26 of ref. 145).    -   influenza antigen(s) (e.g., chapter 17 of ref. 145), such as the        haemagglutinin and/or neuraminidase surface proteins.    -   an antigen from Moraxella catarrhalis (e.g., 153).    -   a protein antigen from Streptococcus agalactiae (group B        streptococcus) (e.g., 154, 155).    -   an antigen from Streptococcus pyogenes (group A streptococcus)        (e.g., 155, 156, 157).    -   an antigen from Staphylococcus aureus (e.g., 158).

The composition may comprise one or more of these further antigens.

Toxic protein antigens may be detoxified where necessary (e.g.,detoxification of pertussis toxin by chemical and/or genetic means (Ref.150)).

Where a diphtheria antigen is included in the composition it ispreferred also to include tetanus antigen and pertussis antigens.Similarly, where a tetanus antigen is included it is preferred also toinclude diphtheria and pertussis antigens. Similarly, where a pertussisantigen is included it is preferred also to includ6 diphtheria andtetanus antigens. DTP combinations are thus preferred.

Antigens in the composition will typically be present at a concentrationof at least 1 g/ml each. In general, the concentration of any givenantigen will be sufficient to elicit an immune response against thatantigen.

As an alternative to using proteins antigens in the immunogeniccompositions of the invention, nucleic acid (preferably DNA, e.g., inthe form of a plasmid) encoding the antigen may be used.

Antigens are preferably adsorbed to an aluminum salt.

Preferred non-streptococcal antigens for inclusion in compositions arethose which protect against Haemophilus influenzae type B (Hib);Typically this will be a Hib capsular saccharide antigen. Saccharideantigens from H. influenzae B are well known.

Advantageously, the Hib saccharide is covalently conjugated to a carrierprotein, in order to enhance its immunogenicity, especially in children.The preparation of polysaccharide conjugates in general, and of the Hibcapsular polysaccharide in particular, is well documented.

The invention may use any suitable Hib conjugate. Suitable carrierproteins are described above, and preferred carriers for Rib saccharidesare CRM197 (HbOC), tetanus toxoid (PRP-T) and the outer membrane complexof N. meningitidis (PRP-OMP).

The saccharide moiety of the conjugate may be a polysaccharide (e.g.,full-length polyribosylribitol phosphate (PRP)), but it is preferred tohydrolyze polysaccharides to form oligosaccharides (e.g., MW from ˜1 to˜5 kDa).

A preferred conjugate comprises a Hib oligosaccharide covalently linkedto CRM197 via an adipic acid linker (Ref. 159, 160). Tetanus toxoid isalso a preferred carrier.

Administration of the Hib antigen preferably results in an anti-PRPantibody concentration of >0.15 μg/ml, and more preferably 1 μg/ml.

Where a composition includes a Hib saccharide antigen, it is preferredthat it does not also include an aluminum hydroxide adjuvant. If thecomposition includes an aluminum phosphate adjuvant then the Hib antigenmay be adsorbed to the adjuvant (Ref. 161) or it may be non-adsorbed(Ref 162). Prevention of adsorption can be achieved by selecting thecorrect pH during antigen/adjuvant mixing, an adjuvant with anappropriate point of zero charge, and an appropriate order of mixing forthe various different antigens in a composition (Ref 163).

Compositions of the invention may comprise more than one Hib antigen.Hib antigens may be lyophilized, e.g., for reconstitution bymeningococcal compositions. Thus a Hib antigen may be packagedseparately from meningococcal conjugates, or may be admixed with them.

Other non-streptococcal antigens for including in compositions of theinvention are those derived from a sexually transmitted disease (STD).Such antigens may provide for prophylaxis or therapy for STDs such asChlamydia, genital herpes, hepatitis (such as HCV), genital warts,gonorrhoeae, syphilis and/or chancroid (Ref. 164). Antigens may bederived from one or more viral or bacterial STDs. Viral STD antigens foruse in the invention may be derived from, for example, HIV, herpessimplex virus (HSV-1 and HSV-2), human papillomavirus (HPV), and/orhepatitis (HCV). Bacterial STD antigens for use in the invention may bederived from, for example, Neisseria gonorrhoeae, Chlamydia trachomatis,Treponema pallidum, Haemophilus ducreyi or E. coli.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, e.g., DNACloning, Volumes I and II (D. N Glover ed. 1985); OligonucleotideSynthesis (M T Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames &S T Higgins eds. 1984); Transcription and Translation (B. D. Hames & S THiggins eds. 1984); Animal Cell Culture (R. I. Freshney ed. 1986);Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A PracticalGuide to Molecular Cloning (1984); the Methods in Enzymology series(Academic Press, Inc.), especially volumes 154 & 155; Gene TransferVectors for Mammalian Cells (J. H. Miller and M. P. Calos eds. 1987,Cold Spring Harbor Laboratory); Mayer and Walker, eds. (1987),Immunochemical Methods in Cell and Molecular Biology (Academic Press,London); Scopes, (1987) Protein Purification: Principles and Practice,Second Edition (Springer-Verlag, N.Y.), Handbook of ExperimentalImmunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds 1986).Remington s Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., 19th Edition (1995); Methods In Enzymology (S. Colowick and N.Kaplan, eds., Academic Press, Inc.); and Handbook of ExperimentalImmunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986,Blackwell Scientific Publications); Sambrook, et al., Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Handbook of Surface andColloidal Chemistry (Birdi. K. S. ed., CRC Press, 1997); Short Protocolsin Molecular Biology, 4th ed. (Ausubel et al. eds., 1999, John Wiley &Sons); Molecular Biology Techniques: An Intensive Laboratory Course,(Ream et al., eds., 1998, Academic Press); PCR (Introduction toBiotechniques Series), 2nd ed. (Newton & Graham eds., 1997, SpringerVerlag); and Peters and Dalrymple, Fields Virology (2d ed), Fields etal. (eds.), B. N. Raven Press, New York, N.Y.

Standard abbreviations for nucleotides and amino acids are used in thisspecification.

All publications, patents, and patent applications cited herein areincorporated in full by reference.

EXAMPLES

To illustrate the methods herein, Streptococcus agalactiae O90. H36b,M781, and CJB111, which produce four representative serotypes (Ia, Ib,III, and V), isolated from patients with GBS disease, were studied.

Example 1 5 and 20 Liter Fermentation

A) Development of Inoculum Preparation Process

The study of the three GBS serotypes' growth was conducted in 5000 mLunbaffled shake flasks containing 1000 mL of the inoculum culture: 8 g/Ldihydrate Na₂HPO₄ (Merck), 2 g/L monohydrate Na₂HPO₄ (Merck), 17 g/Lautolysed yeast extract (Difco laboratories), 1 mg/L biotin (Merck) and33 g/L monohydrate D-glucose (Merck). All compounds were dissolved inreverse osmosis water (ROW) and sterilized by filtration through a 0.22μm pore size membrane filter (Nalgene) and then they were asepticallyadded to the 5000 mL Erlenmeyer flask sterilized in an autoclave at 122°C. for 30 minutes.

For each series of shake experiments, the medium (pH=7.3) was inoculatedwith different volumes of thawed culture (working seed were stored in10% glycerol at −70° C.) and incubated at 35° C. with agitation at 200rpm in a horizontal shaker cabinet (Innova 4330, eccentric 1 inch).

At various times during growth, the optical density at 590 nm wasmeasured (Spectrophotometer novaspecII-Pharmacia Biotech) and pH-valuewas also monitored (pH-meter Metrohm) when the optical density valueswere higher or equal 0.5. The doubling time during the exponential phasewas determined by regression analysis of the linear part of the growthcurve. The slope of the line corresponds to the maximum specific growthrate, μ_(max), and doubling time (t_(d)) according to the formulat_(d)=In2/μ.

B) 5 And 20 Liters Fermentor Preparation

Before filling the vessel and sterilizing the culture medium, thefermentor instruments (pH electrodes and pO₂ meters) were calibratedusing standard methods.

Since GBS is an auxotroph organism, it does not have the capacity tosynthesize particular organic compounds, such as amino acids andvitamins, which are required for its growth. For this reason, a low-costcomplex medium free of components from animal origin was developed, asdescribed in WO07/052168.

The basal medium used for polysaccharide production contained: 2 g/Ldihydrate Na₂HPO₄ (Merck), 16.7 g/L autolysed yeast extract (Difcolaboratories), 32 g/L monohydrate D-glucose (Merck), 1 mg/L biotin(Merck), 0.5 mg/L thiamine hydrochloride (BDH Laboratories), 0.5 mg/Lriboflavin (Fluka), 0.5 mg/L nicotinic acid (Carlo Erba) 0.5 mg/Lpyridoxine hydrochloride (Sigma) and 1 mL·L⁻¹ polypropylene glycol (BDHLaboratories) to prevent foam formation.

Since sterilization of an empty culture vessel was not possible, thefermentor was filled with the phosphate, yeast extract and polypropyleneglycol (4.2 L for a 7 L fermentor and 16 L for a 30 L fermentor) andsterilized in situ at 121° C. for 30 minutes. Some liquid evaporatedduring sterilization. Since the exact loss of liquid due to evaporationwas evaluated empirically with the medium height, a surplus of water wasadded before the sterilization (0.5 L for a 7 L fermentor and 1 L for a30 L fermentor).

In parallel, a concentrated solution of monohydrate D-glucose (550 g/L)and vitamins (0.5 g/L for thiamine hydrochloride, nicotinic acid,pyridoxine hydrochloride. 0.05 g/L for riboflavin and 0.2 g/L forbiotin) were dissolved in ROW, sterilized separately by filtrationthrough a 0.22 μm pore size membrane filter (Nalgene) and thenaseptically added to the fermentation vessel to obtain a right finalconcentration in fermentor (for a 7 L fermentor: 300 mL monohydrateD-glucose, 25 mL biotin, 5 mL vitamin solution (thiamine hydrochloride,nicotinic acid, pyridoxine hydrochloride) and 50 mL riboflavin: for a 30L fermentor: 1000 mL monohydrate D-glucose, 100 mL biotin, 17 mL vitaminsolution and 170 mL riboflavin).

C) Cultivation in 5 L and 20 L fermentors

Expression of cps by GBS, as with others encapsulated bacterialpathogens, is not constitutive but instead varies during growth in vitroand in primary cultures isolated from different sites of infection (Ref.173). Despite such observations, little is known about regulation ofthis surface-expressed carbohydrate antigen in GBS. Cell growth rate incontinuous culture was already reported to be the principal factorregulating capsular polysaccharide production, and growth rate-dependentproduction of type 111 capsular polysaccharide occurred independentlythe growth limiting nutrients. In fact, the production of cps was higherwhen cells were held at a fast mass doubling time (1.4 h⁻¹) than at slow(11 h⁻¹) growth (Ref. 174).

Initially, all studies with GBS were performed with cells grown in batchculture, which were characterized by changing growth rate, nutrientsconcentration, and pH. Growth parameter shifts experienced by cells inbatch culture lead to metabolic changes that affect the composition ofthe cell. Continuous culture allows for continuous exponential growth inan environment of stable substrate, product, and biomass concentrationsand at a rate controlled by the researcher. If growth rate conditionsare maintained, a steady state will be achieved. However, continuousculture should be avoided for industrial production because it is proneto strain stability problems and contamination, and is also expensive onmanufacturing-scale due to the need for a continuous feed of medium andnutrients.

Cultivation at the 5 L- and 20 L-scale was carried out respectively in aBiostat CT5-2 and C20-3 reactors (B. Braun Biotech International), whichhad a total volume of 6.9 L and 31 L. The bioreactors were respectivelyequipped with 2 and 3 stirrers, each containing six paddles. Inaddition, ports for steam-sterilizable probes to measure the dissolvedoxygen concentration (Inpro 6500 series2 oxygen sensor; Mettler Toledo),pH (model Pa/2; Mettler Toledo), temperature (pt100 electrode, M. K.Juchleim GmbH), foam (model L300/Rd.28; B. Braun Biotech International)were available. The operations were controlled and recorded with a DCU-3digital controlled unit in combination with the MFCs/win softwarepackage (B. Braun Biotech Intemational). Carbon oxygen and oxygenconcentrations in the spent gas leaving the bioreactor were measuredwith 1310 fermentor monitor (Innova) and a GMUX-8 analyzer (B. BraunBiotech International).

Cultivation was done at 36° C.±1° C. with 0.2 bar of pressure andpO₂=30±10% saturation in the medium, which was controlled by agitationrates, between 100±10% and 700±10% rpm for a 7 L fermentor, and between50±10% and 500±10% rpm for a 30 L fermentor, and by aeration rates of0.1 and 1.0 v/v/m±10%. The pH of the medium was kept at 73±0.2automatically by controlled addition of 4 M hydroxide sodium. Themonitoring and/or control of various parameters such as temperature, pHand agitation were performed in a PID control unit. Foam was controlledautomatically with an antifoam agent emulsion (BDH Laboratories).

1000 mL of the inoculum sterile medium was inoculated with an adaptedworking seed volume of the strain studied and incubated for the desiredtime at 35° C. with shaking on a rotary shaker. When the inoculum flasksreached mid-exponential phase (OD between 0.8 and 1.2), sufficientvolume of this culture was used to inoculate fresh batch medium (4.7 Lor 17 L) to result in an initial OD of 0.032.

The batch phase of fermentation was allowed to proceed until the cultureOD_(590nm) reached 2.5 (±0.5). When the culture was at or near this ODvalue, the first exponential fed batch addition of yeast extract medium(150 g/L) was initiated and continued for 45-50 minutes to maintain aspecific growth rate (p) of 0.138 h⁻¹. A second exponential fed batchaddition of yeast extract media (150 g/L) to maintain a μ of 0.924h⁺⁺⁺was initiated at the end of the first part when the OD was equal to4.5±0.5 and continued for 45-50 minutes. These addition phases reducedthe bacteria's doubling time from 20-25 minutes, which is typical of thebatch phase, to 45 minutes which allowed the micro-organism to adapt tothe ideal conditions for polysaccharide production. Ultimately, toincrease productivity, at the end of the second addition of yeastextract when the OD was at 10.0±2, the culture was continued using apH-regulated feed of concentrated monohydrate D-glucose (550 g/L) for 3hours, avoiding completely depletion of the substrate which would resultin pigment production and a reduction of capsular polysaccharideproduction.

Small samples of 50 mL were withdrawn from the culture fluid atintervals during the fermentation processes and analyzed for bacterialgrowth, glucose consumption and polysaccharide production.

D) Harvest and Inactivation of GBS

The culture was harvested when the growth rate consistently slowed down,which occurred 3 hours after the initiation of the pH-controlled glucosephase. The culture was immediately centrifuged at 7741×g for 45 minutesat ambient temperature (Avanti™ centrifuge J-20 XPI Beckmam coulter).The supernatant was removed and conserved at −20° C. for glucose assays,and the weight of harvest was determined.

Purification of GBS polysaccharides first required the inactivation andhydrolysis of cps. 1 M sodium hydroxide was added to the pellet toobtain a final concentration of 0.8 M. The reaction mixture wasincubated for 36 hours at 37° C. and 120 rpm before the serotypespecific cps content was determined.

E) Fermentor Cleaning

After the culture was harvested, the vessel and accessories weredecontaminated. First, the fermentors were filled with ROW and the pHwas increased to 12 by manually addition of 4 M sodium hydroxide.Sanitization was performed by heating the water to 80° C. for 30minutes, maintaining a pressure of 0.2 bar and an agitation of 200 rpmthat ensured a homogenous dispersion of heat. When the sanitization wascompleted, the water with sodium hydroxide was harvested, and thefermentor was washed with ROW until the pH-value was decreased to arange 5-7. Generally, three washings were necessary before reaching thedesired pH. The fermentor was emptied, and the probes and accessorieswere removed from their ports. The septum connectors for inoculation andtransfer of additive nutrients and corrective agents, and the bottlesfor removal and storage samples were sterilized separately in anautoclave for 30 minutes at 122° C. The fermentor was again filled withROW, and sterilized at 121° C. for 30 minutes.

Example 2 2 Liter Fermentation

A) Strain and Cultivation Medium

Streptococcus agalactiae type III strain M781, originally isolated froma newborn with GBS meningitis, was provided by Carol J. Baker. StrainM781 cells were grown in a modified version of a chemically definedmedium, initially developed for group A streptococci (Ref. 174). Thecomposition of the chemically defined medium used in the batch culturestudy is listed in Table I.

TABLE 1 Chemically defined medium composition batch phase FINAL CHEMICALCONCENTRATION COMPOUNDS (mg · L⁻¹) PRODUCER Carbon source in ROWD-Glucose•H₂O 20000 Merck Phosphate solution in ROW K₂HPO₄ 300 MerckKH₂PO₄ 440 Merck Na₂HPO₄•2H₂O 3150 Merck NaH₂PO₄•H₂O 2050 Merck NaCl 10Merck Sulphate solution in ROW (NH₄)₂SO₄ 600 Ashland MgSO₄•7H₂O 200Merck MnSO₄•H₂O 10 Sigma FeSO₄•7H₂O 10 Sigma Sodium solution in ROWSodium citrate 225 Sigma Sodium acetate 6000 Carlo Erba Vitaminssolution in ROW Biotin 0.01 Merck Nicotinamide 2 Carlo Erba CaPanthotenate 0.8 Merck Riboflavin 0.4 Fluka Thiamine hydrochloride 0.4Merck Pyridoxine hydrochloride 0.8 Sigma Vitamin solution in sodiumhydroxide 1 mol · L⁻¹ Folic acid 0.1 Sigma Nitrogenous bases in sodiumhydroxide 1 mol · L⁻¹ Adenine 35 Sigma Guanine 27 Sigma Uracil 30 SigmaAmino-acids in ROW L-Alanine 200 Sigma L-Arginine 200 Sigma L-Glutamine5 Sigma Glycine 200 Sigma L-Histidine 200 Sigma L-Isoleucine 100 SigmaL-Leucine 100 Sigma L-Lysine 110 Sigma DL-Methionine 100 SigmaL-Phenylalanine 100 Sigma L-Proline 200 Sigma DL-Serine 100 SigmaL-Threonine 100 Sigma L-Tryptophan 200 Sigma L-Valine 100 SigmaAmino-acids in sodium hydroxide 1 mol · L⁻¹ L-Aspartic acid 100 SigmaL-Cysteine hydrochloride 200 Sigma L-Glutamic acid 300 Sigma L-Tyrosine200 Sigma

The pH probe was calibrated by a two point calibration using twostandard solutions (pH-values=7 and 4). The pO₂ and pH probes weremounted in the culture vessel. The fermentor (Applikon 3 L) was filledwith glucose and sterilized in an autoclave at 122° C. for 30 minutes.All others compounds were sterilized separately by filtration through a0.22 μm pore size membrane filter, and then aseptically added to thefermentation vessel. In order to avoid the precipitation of medium,concentrated solution were prepared (20× phosphate, 100× sulphate, 50×sodium, 240× nitrogenous bases, 9000× vitamins, 35× amino acids in waterand 90× amino acids in sodium hydroxide) and an appropriate volume ofeach was added in the following order to the carbon source to producethe desired final concentration: phosphate, amino acids, vitamins,sulphate and nitrogenous bases. When all additions were performed, thepO₂ probe was calibrated. For the “zero” measurement, the culture vesselwas sparged overnight with nitrogen. After the culture was saturatedwith oxygen and when the operations conditions were reached theelectrode slope was calibrated to 100%.

B) Cultivation at 2 L Scale

Preliminary experiments on the expression of capsular polysaccharides bystrain M781 were performed in batch culture.

For cell activation, 100 mL of the chemically defined medium (pHvalue=7.2, adjusted using 6 M chlorhydric acid), sterilized byfiltration though a 0.221 m pore size membrane filter (Nalgene) andplaced in sterile 500 mL Erlenmeyer flasks, were inoculated with 0.1 mLof thawed culture of M781 strain (working seed were stored in 10%glycerol at −70° C.). This seed culture was incubated at 35° C., withagitation at 200 rpm in a horizontal shaker cabinet (Innova 4330,eccentric 1 inch) for 9 hours. The seed culture was then inoculated into1.8 L of the basal medium in a 2.5 L jacketed fermentor (Applikon),whereby the initial OD was 0.4.

The fermentation was controlled by a digital measurement and a controlunit from Applikon Instruments (Biol controller ADI 1030, Applikon), andall data were collected by computer (BioXpert software). The temperaturewas automatically controlled at 36° C. by an external thermostat(Haake). Dissolved O₂ was measured by a sterilizable electrode(Applisens) and was maintained above 30% of air saturation by automaticadjustment of the agitation speed between 150 to 1000 rpm (MotorController ADI 1012) and aeration rates of 0.1 and 1.0 v/v/m (Flowconsole Applikon). The culture pH was maintained at 7.3 by automatictitration with 2 M sodium hydroxide (pump driver Masterflex).

For the fed batch culture studies with unknown growth limiting factors,the cultivation was initiated with a batch growth phase (1.2 L),followed by a feeding phase. To develop the best fed batch strategy,three fed batch techniques were tested for each cell growth: (1)pH-stat, (2) DOT-stat, and (3) exponential. The cps concentration wasmonitored at regular intervals.

To control substrate feeding using the pH-stat method, the cultivatingmedium's pH was adjusted to 7.3 during the cultivation processes by theaddition of a feed solution with a peristaltic pump (Masterflex ConcodeDrive). When the pH exceeded the set point of 7.3, glucose was depleted.Consequently, supplemental nutrients were automatically added toreadjust the pH to the set point.

For the pO₂-stat strategy, the carbon source feed was addedautomatically whenever the pO₂ increased above the set point of 40% ofdissolved oxygen.

For the exponential cultivation technique, the peristaltic pump wasactivated at the end of exponential phase to maintain a specific growthrate equal to 0.92h⁻¹ (t_(d)=45 minutes), the optimal doubling time forcapsular polysaccharide production. Feeding was accomplished accordingto the following formula:

$F = \frac{{\mu ( {V\; X} )}_{0}^{utf}}{S \cdot Y_{X/S_{f,{const}}}}$

where F is the feeding rate, g is the specific growth rate, (VX)₀ is thebiomass at the start of feeding, if is the time when feeding started,S_(f,const) is the substrate concentration of feed, and Y_(X/S) is thecell yield coefficient for glucose.

-   -   C) Shake Flasks Study to Identify the Growth Factors        Requirements

Growth factor requirements of the organism were determined byeliminating individual nutrients from the defined medium and bydetermining whether the resultant medium would support growth. The studywas conducted in 500 mL unbaffled shake flasks containing 100 mL ofchemically defined medium (see Table 1).

The pH of the cultivating medium was adjusted to 7.2 using 6 Mchlorhydric acid. The cultivating medium was then sterilized byfiltration though a 0.22 μm pore size membrane filter (Nalgene). Foreach series of shake experiments, the cultivating medium was inoculatedwith 0.1 mL of thawed culture of M781 strain (working seeds were storedin 10% glycerol at −70° C.), and incubated at 35° C. with agitation at200 rpm in a horizontal shaker cabinet (Innova 4330, eccentric 1 inch).After 18 hours of cultivating, the OD at 590 nm (Spectrophotometernovaspecll-Pharmacia Biotech) and pH-value (pH-meter Metrohm) weremeasured.

The growth in amino acid and vitamin deficient media was compared with acontrol culture with all compounds present.

Example 3 Analytical Methods

A) Growth Measurements

As soon as the samples were collected, the biomass content was monitoredby reading the OD of the culture at a wavelength 590 nm (Novaspec IIspectrophotometer—Pharmacia bioteck). Dilutions of the samples wererealized in order to read a value of absorbance within the interval0.10-0.50.

Cell concentration, defined as g/L of chemically defined medium wasdetermined by placing an accurately measured volume of culture broth (30mL) into a previously dried and weighed 50 mL polypropylene centrifugetube. Cells were centrifuged at 27,217×g for 45 minutes at 4° C. in anAvanti-TM JA-20 XPI Beckman Coulter refrigerated centrifuge. Thesupernatant was decanted and the cell pellet was dried in an oven at 85°C. for 24 hours, and weighed. A relationship between the OD at 590 nmand g_(CDW)/L (cell dry weight) biomass was established. An OD of 1 at590 nm was equivalent to 0.44 g_(CDW)/L biomass.

-   -   B) Gram Staining

Gram's stain differentiates between two major cell wall types. Bacterialspecies with walls containing small amounts of peptidoglycan and,characteristically, lipopolysaccharide, are Gram-negative, whereasbacteria with walls containing relatively large amounts of peptidoglycanand no lipopolysaccharide are Gram-positive. This method, used for bothlaboratory and pilot scale, provided assurance that the seed culture andfermentor culture were pure.

With respect to the staining technique, cells on a microscope slide wereheat-fixed and stained with a basic dye, crystal violet, which stainsall bacterial cells blue. Then, the cells were treated with aniodine-potassium iodide solution that allowed the iodine to enter thecells and form a water-insoluble complex with the crystal violet dye.The cells were treated with an alcohol solvent, in which theiodine-crystal violet complex was soluble. Following the solventtreatment, only gram-positive cells remained stained.

After the staining procedure, cells were treated with a counterstain,safranin to visualize the decolorized gram-negative cells.Counterstained gram-negative cells appeared red, while gram-positivecells remained blue. After the counterstain was rinsed off, the slidewas gently warmed to remove any residual moisture. The slide was thenplaced on a microscope stage, where the oil-immersion lens was loweredinto the immersion oil.

C) Glucose Assays

In culture supernatants, glucose consumption was determinedcolorimetrically by measuring the absorbance of the solution in a 1 cmlight path at 340 nm (NADPH) and by comparing it to a standard curveprepared by assaying pure glucose.

D-Glucose was phosphorylated to D-GIucose-6-phosphate in the presence ofthe enzyme hexokinase and ATP with the simultaneous formation of ADP:

In the presence of the enzyme glucose-6-phosphate dehydrogenase.D-Glucose-6-phosphate was oxidized by NADP to D-gluconate-6-phosphatewith the formation of NADPH:

The amount of NADPH formed in this reaction was stoichiometric to theamount of D-Glucose.

The amount of D-Glucose present in the assay was between 1 μg and 50 μg.In order to get a sufficient absorbance difference, the sample solutionwas diluted to yield a D-Glucose concentration between 0.08 and 0.5 g/L.

The supernatant, stocked in fridge at −20° C. to stop enzymaticreaction, was serially diluted in ROW. 50 μL of diluted supernatant wasincubated at ambient temperature for 15 minutes with 10 μL of a solutioncontaining hexokinase (approximately 320U) and glucose-6-phosphatedehydrogenase (approximately 160U) in triethanolamine buffer with NADP,ATP and magnesium sulphate (pH=7.6).

After preparation of the sample in the disposable cuvette (1 cm lightpath) according the Roche procedure, the spectrophotometric measurementwas performed at room temperature at 340 nm against air.

D) Determination of Capsular Polysaccharide Content

N-acetyl-D-neuraminic acid (sialic acid) is an acidic sugar frequentlyfound as a component of eukaryotic carbohydrate structures(glycoproteins and glycolipids). In prokaryotic cells, sialic acid hasalso been found as a constituent cps of a few genera of pathogenicbacteria [10]. In fact, the serotype-specific cps of GBS comprises arepeating unit of the following saccharides: N-acetyl-neuramic acid orsialic acid, glucose, galactose and N-acetylglucosamine. Since sialicacid is an integral component of the polysaccharide, its quantitativedetermination was used to monitor serotype specific cps productionfollowing the chemical method setup by Svennerholm (Ref. 176).

Before determining the quantity of sialic acid, the cps in theinactivated sample was partially purified and concentrated. After thehydrolysis of polysaccharides in sodium hydroxide for 36 hours, 1.5 mLof the inactivated samples was centrifuged at 15600×g for 25 minutes(Centrifuge 5415R—Eppendorf) to remove the cells. The supernatant wasdiluted in ROW at 1:10, and then purified and concentrated using acentricon centrifugal filter (Millipore's Ultracel YM-30 regeneratecellulose). Concentration was achieved by utrafiltering the dilutedsample through an anisotropic membrane according to the followingprocedure.

After inserting the sample reservoir into a filtrate vial, 1.5 mL ofwater was added twice to a sample reservoir, and spun for 20 minutes at2519×g and 20° C. to clean the centricon system. Once the system wasready, 1.5 mL of the diluted samples were added and centrifuged at2519×g for 30 minutes. The centrifugal force drove solvents and lowmolecular weight solutes through the membrane into the filtrate vial.Macromolecules such as the cps were retained above the membrane insidethe sample reservoir. As the sample volume was diminished, the retainedsolute concentration increased. The retentate was washed three timeswith 0.5 mL of NaCl 0.5 M to eliminate the contaminants, and wascentrifuged at 2519×g at 20° C. for 20 minutes.

For recovery, 0.5 mL of 0.5 moI·L⁻¹ NaCl was added to the samplereservoir. The cps was transferred to the retentate vial by placing thevial over the sample reservoir, inverting the device, then centrifugingfor 1 minute at 205×g. This recovery process was repeated twice torecover all the cps.

To quantify the amount of sialic acid, a colorimetric method involvingresorcinol-hydrochloric acid was used. The reagent for the quantitativeassay contained 0.2 g of resorcinol (Merck) in a solution containing 80mL of 37% HCl to assure acid hydrolysis. 20 mL of water and 20 μmol ofCuSO₄5H₂O (Merck).

The assay was performed as follow: 1 mL of the sample containing 5-25 μgof sialic acid was added to 2 mL of resorcinol reagent. After theaddition of reagent, the solutions were mixed and heated at 100° C. for20 minutes in a boiling water bath, during which a blue-violet colordeveloped.

The tubes were cooled at room temperature, and the absorbance was readat 554 nm using a Novaspec 11 spectrophotometer. Disposable cuvetteswith a 1 cm light path and 1 mL capacity were used. The absorbance wasdirectly proportional to the concentration in the range 5-25 μg ofsialic acid.

In order to compensate for non-specific color in biological materials, acontrol sample was run without resorcinol. The sample blank absorbancewas subtracted from the test sample before calculating the amount ofsialic acid.

In parallel, standard solutions of 0, 5, 10.15, 20 and 25 gN-acetylsialic acid were prepared under the same conditions. The amountof N-acetylsialic acid was calculated using the equations derived fromthe standard solutions of N-acetylsialic acid with the program UVLAMBDA.

Capsular polysaccharide of O90. H36b and M781 contains 31% (w/w)N-acetylsialic acid. The capsular polysaccharide of CJB111 contains 23%(w/w) N-acetylsialic acid. Thus, the cps concentration could beascertained using this correction factor. Volumetric production of cpswas expressed in units of mg/L, and specific cps production wasexpressed in units of mg/L·OD.

Example 4 Simplified Complex Media and Linear Additions

A) Development of Inoculum Preparation Process

In the fermentation process to produce serotype specific cps of GBS,2000 mL shake flasks containing 500 mL of medium were used to preparethe inoculum culture. However, these flasks were not suitable for pilotand production scale. Thus, the behavior of four GBS strains (O90, H36b,M781, and CJB111) was studied in new flasks to develop an inoculumpreparation process suitable for pilot scale. This procedure was thenused to produce pre-phase I clinical lots according to cGMP.

The first goal of this example was to find the growth parameters ofthese four GBS strains in 5000 mL Erlenmeyer flasks containing 1000 mLof medium, and to also study the optimal culture time required for alate-exponential growth phase with a suitable pH-value in the fermentor.The second goal was to study the volume of working seeds used toinoculate the flasks in order to modify the culture time such that itcould be initiated either the evening before the fermentation day(culture time about 8 hours) or early in the morning of the fermentationday (culture time about 3 hours), as appropriate for the growth rate ofthe strain.

The study of the three GBS serotypes was conducted using 3 mL of theworking seed to inoculate 1000 mL of fresh sterile inoculum medium.Furthermore, each experiment was performed twice to ensurereproducibility. However, since the growth curve and doubling timeobtained were similar, only one result of each experiment has beenpresented.

For the O90 strain, the cells remained in the exponential growth phasefor 6.5 hours with a doubling time of 30 minutes (μ_(max)=1.41 h⁻¹).Therefore, 6 hours was sufficient to attain the mid-exponential growthphase using a suitable pH value of 6.8). Since this length of time wasnot enough to inoculate the flasks in the evening before thefermentation day, the working seed volume was reduced to 0.1 mL toincrease this cultivation time. Under these conditions, the bacteriaremained in exponential growth phase for 8.5 hours with a doubling timeof 35 minutes (μ_(max)=1.20 h⁻¹). Thus, the preparation of the inoculumculture for this strain required 8 hours.

For the H36b strain, only one experiment with 3 mL of the working seedwas performed. The cells immediately entered exponential growth phasewith a doubling time of 25 minutes (μ_(max)=1.65 h⁻¹), and thedeceleration phase was initiated after 4.5 hours. Thus, in order toobtain an OD in the range 0.8-1.2 with pH-value around 6.5 (7+0.5), themedium for this strain had to be inoculated 4 hours before fermentation.

For the M781 strain, the experiment was performed twice, first using aworking seed volume of 3 mL and then using 0.1 mL. Using 3 mL of theworking seed volume, 7.5 hours was necessary for the cultivation time.In order to increase the culture time by 2.5 hours to allow culturingovernight (5 doubling times), 0.1 mL of working seed volume was used. Itwas observed that the difference time was only 30 minutes and doublingtime was reduced from 31.4 minutes (μ_(max)=1.32 h⁻¹) to 27.5 minutes(μ_(max)=1.51 h⁻¹). Thus, using 0.1 mL of working seed volume for thisstrain, the flasks had to be inoculated for 8 hours before fermentation.

For the CJB111 strain only one experiment with 3 ml of the working seedwas performed. The cell immediately entered in exponential growth phasewith doubling time of 21 min (μ_(max)=1.98 h⁻¹) and the decelerationphase began after 4.5 h. Thus, in order to obtain an OD in the range of0.8-1.2 with a pH-value around 6.5 (7.0+/−0.5), the medium had to beinoculated 4 hours before fermentation.

Before this new inoculum process could be transferred to pilot scale,the behavior of the four GBS strains was studied in cGMP conditions tovalidate the inoculum preparation process. As reported in GoodManufacturing Practices (volume 4): “Validation studies were conductedin accordance with defined procedures. When any new manufacturingformula or method of preparation was adopted, steps should be taken todemonstrate its suitability for routine processing. The defined process,using the materials and equipment specified, should be shown to yield aproduct consistently of the required quality.” The new inoculumpreparation process was repeated using the optimal working seed volumeof each strain. The growth behavior was identical to the precedingstudy. For the O90 strain, the working seed volume was 0.1 mL, and theculture time was 8.50 hours to achieve the desired range whereby the ODis 1±0.2 and pH is 6.5±0.2.

For the H36b strain, the working seed volume was 3 mL, and the culturetime was 4 hours to achieve the late exponential phase. For the M781strain, the working seed volume was 0.1 mL, and the culture time was7.50 hours to achieve the desired OD range of 0.8-1.2. For the CJB111strain, the working seed volume was 3 mL and the culture time was 4hours to achieve the desired OD.

This inoculum preparation process was used in pilot scale, and required4 flasks containing 1000 mL of medium to achieve an initial OD of 0.032in a 200 L fermentor.

B) Development of Fermentation Process

i) Verification of the Need to Add Vitamin Solutions in the ComplexCultivating Medium

Since GBS is an auxotroph organism, it does not have the capacity tosynthesize particular organic compounds, such as amino acids andvitamins, which are required for its growth. For this reason, a low-costcomplex medium free of components from animal origin was developed, asdescribed in WO07/052168. This complex cultivating medium was sterilizedby heat, to which biotin and vitamin solutions of thiamine, riboflavin,nicotinic acid and pyridoxine at 0.5 g/L in 0.1 M sodium hydroxide andmethanol were added.

The yeast extract used for the growth of the GBS strains contained thesevitamins, even if the ratio may differ significantly depending uponproduction process and processing of yeast autolysates (Table 2) (Ref.177).

TABLE 2 Vitamin physiological meanings of GBS and concentrations inyeast extract Vitamin concentration (mg/L) Vitamin in the medium due toconcentration (mg/L) the addition of the in the medium due toPhysiological role vitamin solution the yeast extract Vitamin B1:Essential component 0.5 1.08 Thiamine of a thiamine pyrophosphatecoenzyme involved in energy metabolism Vitamin B2: Role in oxido- 0.54.28 Riboflavin reduction reaction Vitamin B3: Electron carrier in 0.524.5 Nicotinic Acid dehydrogenation reaction Vitamin B6: Coenzyme in 0.50.83 Pyridoxine transamination reactions involving α- amino acidsVitamin B8: Biotin Importance in fatty 1.0 0.09 acids, amino acids andcarbohydrate metabolism

To attain a simple cost-effective production process, the role of biotinand the four vitamin solutions added to the complex cultivating mediumwere assessed for their effect upon growth and cps production of thethree specific strains of Streptococcus agalactiae.

Before developing the fermentation process to transfer of the cpsprocess to pilot scale, the growth of the three GBS serotypes wasmonitored in a fermentor at laboratory scale using parameters developedin previous Examples.

In these conditions, final ODs after 3 hours of glucose feed varied from14 for the O90 strain to 28.5 for the H36b strain, and the cpsconcentration for these strains was between 300 mg/L and 550 mg/L.

Since methanol had to be avoided for safety reasons and vitamins insodium hydroxide lost their property, a ROW solution of thiamine,pyridoxine and nicotinic acid was selected. At the same time, a secondmodification was made to the process. Since vitamins are thermo-labilecompounds, the yeast extract medium was sterilized by filtration througha 0.22 μm pore size membrane filter rather than autoclaving, andaseptically added to the phosphate medium, rather than by sterilizationusing an autoclave at 121° C. for 30 minutes.

A new fermentation experiment was performed for each strain taking intoconsideration the modifications previously described. Although thegrowth for the three stains was equal (H36b) or better (O90 and M781)than the process established in previous Examples, the pigmentation ofthe culture persisted. In fact, in an article by Fraile et al., theproduction of an orange-yellow pigment integrated in the cell wall was aspecific characteristic of human haemolytic GBS and served as the basicfor use of culture media to identify GBS from clinical samples (Ref.178). In order to eliminate this chemical contamination (color), thepurification process required an additional step using a Z-carbonsurface.

With respect to the cps production at the end of fermentation, themodifications increased cps concentration by about 100 mg/L for the O90and H36b strains and 300 mg/L for the M781 strain. Furthermore, thepercentage of cps by gram of cell dried weight was higher. The additionof riboflavin was not necessary for these three strains of Streptococcusagalactiae because the vitamin concentration in the yeast extract wassufficient to satisfy the nutritional needs of these strains.

In order to reduce vitamin solutions added to the medium, experimentsonly using biotin were performed. Although the cps concentration wasslightly decreased for the H36b and M781 strains, the cps production wasstill an improvement over the process established in previous Examples.The addition of vitamins to the complex cultivating medium was notnecessary for the growth and cps production of Streptococcus agalactiaebecause the vitamins in yeast extract were sufficient to satisfy thenutritional needs of the three specific strains of GBS with theexception of biotin.

Moreover, according to the purification data, the cps structure in theabsence of vitamins was comparable to the structure obtained in theprocess established in previous Examples, the acetylation was low, andthe purity of product was acceptable.

The growth of the O90 strain was also monitored upon removing biotin. Inthis experiment, both the OD decreased from 18 to 14 in presence ofbiotin and the cps concentration was reduced by more than 100 mg/L. Thebiotin concentration in the yeast extract was not sufficient to satisfythe growth needs of this GBS strain. In fact, according to yeast extractcomposition, biotin was present in lower quantities (0.25 mg/100 g) whencompared to the other vitamin concentrations.

In conclusion, removal of thiamine, riboflavin, nicotinic acid andpyridoxine had little effect on the growth and cps production of GBS.For theses reasons, the four vitamins were removed from the fermentationprocess, so that only biotin was added to the complex cultivatingmedium. For CJB111, the final conditions using biotin only was used.

ii) Verification of the Necessity of Complex Fed Batch Process toProduce Serotype Specific Capsular Polysaccharides of GBS

Cell growth rate was previously reported to be the principal factorregulating cps production, and the growth rate-dependant production oftype III cps occurred independently of the growth-limiting compounds(Ref. 173). However, the depletion of carbon source was found to be acause of pigment formation and reduction of capsular polysaccharideproduction. To maintain a nutritious environment and a growth ratefavorable to cps production, a complex fed batch fermentation processwas developed as described in WO07/052168. This complex fed batchprocess combined both an exponential technique to reduce the bacteria'doubling time and a pH-stat technique with glucose in the last 3 hoursto increase cps productivity. This process combined the advantages ofbatch and continuous techniques. In fact, fed batch fermentationachieves high cell densities by extending the exponential growth phaseand control over substrate addition conditions during fermentation.However, the use of a complex fed batch technique requires usingsoftware that manages the fermentation through algorithm, and use ofthis software necessitates the validation of the algorithm to complywith GMP standards. Therefore, the fermentation process was simplifiedto avoid using the algorithm.

In accordance with the process established in previous Examples, thesame OD values were used as triggers for initiation of each feed and theinstantaneous additions. Furthermore, 150 g/L of yeast extract and 500g/L of glucose were added to the batch, which constituted 10% of initialbatch volume. The two instantaneous additions of yeast extract at OD of3 and 4.5, respectively, constituted ⅕th and ⅘th of the total requiredvolume. When the OD reached 10, a linear addition of concentratedglucose was initiated to replace the pH-stat phase. The velocity of thisaddition was calculated to add the same amount of glucose as the complexfed batch process in 3 hours.

The fermentation was performed for each strain using the new process,and the cell density and cps production were monitored. For the O90strain, the simplified process produced the same result as the complexfed batch process. For both the O90 and H36b strains, the growth wasfaster than complex fed batch technique and the OD at the end of theprocess increased from 24 to 32. The cps concentration and the cpsquantity by gram of cell dried weight increased by approximately 300mg/L and 5 mg_(cps)/g_(CDW), respectively. For the M781 strain, the samegrowth and cps production was observed. Thus, the fermentation processcould be simplified using the linear addition of glucose without pHmonitoring. For CJB111, the only the final process with the simplifiedlinear addition without pH monitoring was performed to verify theefficacy of the protocol.

This process using two instantaneous additions of yeast extract and alinear addition of glucose was the preferred pilot-scale method. The newprocess did not require the use of an algorithm or the addition ofvitamin solutions to the cultivating medium. The complex cultivatingmedium, which was comprised of yeast extract, phosphate, glucose andbiotin, was a low-cost robust process that led to reproducible growthbehavior and cps production.

C) Pre-Validation of Fermentation Process

The previously developed fermentation process to produce cps of GBS wasvalidated and optimized. The growth and cps production were monitoredfor the H36b strain, whereby each parameter was modified individuallyand compared with a control culture. The DOT study, the temperature andpH were reported.

The cultivation was performed at 36° C. with a pH of 7.3, and thedissolved oxygen in the medium was maintained at 30% during the entireprocess. After 3 hours of feeding glucose, the final OD was 25.3, andthe average productivity was 1.84 g/L·h. The cps concentration andquantity of cps in one gram of cell dried weight were respectively 540mg/L and 59 mg_(cps)/g_(CDW).

i) Effect of the Dissolved Oxygen Level

Streptococcus agalactiae is a facultative anaerobic organism thatsynthesizes ATP by aerobic respiration if oxygen is present; however, itis also able to switch it to anaerobic growth.

First, the dissolved oxygen in the medium was maintained at 15%. Theaverage productivity was reduced from 1.84 g/L·h to 1.14 g/L·h but thesame OD of 23.2 at 590 nm was observed at the end of the process. Thecps concentration was lower at around 406 mg/L and less cps wereproduced by gram of cell dry weight (39.8 mg_(cps)/g_(CDW)).

The same fermentation was performed maintaining the dissolved oxygen at60%. In this case, the average productivity was 1.16 g/L·h and the finalOD was 20.5. The specific productivity was decreased to 433 mg/L and thequantity of cps by gram of cell dry weight was reduced to 49mg_(cps)/g_(CDW).

In light of these observations, 30% of dissolved oxygen was selected forthe pilot-scale production, using an agitation between 50-350 rpm andair flow between 20-100 L/min. Oxygen, which is expensive as a gas, wasonly used in the last hour of the fermentation process, thereby keepingthe cost of the manufacturing process down.

ii) Effect of the Temperature

The growth and cps production were also monitored by modifying thetemperature by increasing and decreasing the temperature by 2° C. withrespect to the standard temperature at 36° C.

By lowering the temperature to 34° C., the doubling time was reduced andthe average productivity was decreased from 1.84 g/L·h to 1.04 g/L·h.However, the cps concentration was decreased to 60% (312 mg/L), andapproximately 20 mg_(cps)/g_(CDW) were lost at this temperature.

When the process was repeated at 38° C., a reduction in averageproductivity was observed from 1.84 g/L·h to 1.45 g/L·h. Furthermore, asignificant reduction of both cps concentration and the quantity of cpsper gram of cell dry weight was observed at the end of the process (267mg/L and 32.1 mg_(cps)/g_(CDW), respectively).

Since modifying the temperature of the fermentation process affected theGBS doubling time and considerably reduced serotype specific cpsproduction, 36° C. was confirmed to be optimal temperature for GBSgrowth and cps production.

iii) Study of the pH-Values

Experiments were also performed to optimize the pH-value, by varying theoriginal pH at 7.3 to 7.0 and 7.5. When the pH was maintained at 7.0,the final OD was increased from 23.5 to 28.8, and the averageproductivity was 1.52 g/L·h. However, cps concentration was decreasedfrom 540 mg/L to 412mg/L.

The same fermentation process was performed with a pH of 7.5. The samegrowth behavior for OD, but a average productivity of 1.08 g/L·h wasobserved and a significant decrease of cps volumetric productivity wasnoted from 540 mg/L to 323 mg/L. Thus, maintaining a pH of 7.3 duringthe fermentation process was optimal for cell density and cpsproduction.

iii) Study of the Pressure-Values

Experiments were also performed to optimize the pressure by comparingthe fermentation process at two pressures: 0.2 to 0.5 bar. When thepressure was maintained at 0.5, the final OD was maintained 23.5, andthe average productivity was 1.5 g/L·h. However, cps concentration wasdecreased from 540 mg/L to 272 mg/L.

After studying the effects of dissolved oxygen, temperature, pH, andpressure, conditions previously established were found to be the optimalconditions for producing both high cell density and serotype specificcps.

Example 4 Development of a Chemically Defined Medium

Since, the complex cultivating medium used in preceding Examplescontained organic sources whose compositions are not completely known(e.g., yeast extract), variability in the performance of thefermentation process was observed. One approach to reduce variabilitywhile maintaining productivity is to replace the complex medium with achemically defined medium which primarily consists of inorganiccompounds. This replacement allows the fermentation process to becontrolled, and also simplifies the purification of polysaccharides.

Previous studies (Ref. 179) demonstrated that Streptococcus agalactiaecould be grown in a chemically defined medium which supported a rate andan amount of growth comparable to that obtained in the complex medium.The purpose of this investigation was first to study the growthcharacteristics and examine growth factors requirements of M781 strainof Streptococcus agalactiae representing serotype III. To increase theyield of biomass and cps production, a simple fed batch process wasdeveloped.

A) GBS Growth Study in a Chemically Defined Medium

To develop a fed batch process, typically the micro-organism must firstbe analyzed to ascertain the best abiotic conditions, the differentgrowth phases, the consumed and produced components, the relationshipbetween the biomass and product formation, the limiting substrate forgrowth and the relationship between the specific growth rate and thelimiting substrate concentration. However, behavioral information aboutGBS was already known from the studies performed to develop the complexcultivating medium. The optimum conditions for pH and temperaturedeveloped in preceding Examples for GBS growth in the complexcultivating medium were extended to the chemically defined medium as setforth in Table 1 above.

i) GBS Growth Stud in Erlenmeyer Flasks

Preliminary experiments on the growth by strain M781 were performed inbatch culture using 500 mL Erlenmeyer flasks. The cells were maintainedin exponential phase for 8 hours with a doubling time of 45 min(μ_(max)=0.91 h⁻¹). After this first growth phase, the specific growthrate began to decrease and the cells were in deceleration phase for 2hours. The final optical density was around 1.5 and the exponentialphase was finished when optical density was around 0.7.

ii) GBS Growth Study in 2 L Fermentor

Next, GBS growth was monitored in a 2 L fermentor. Under theseconditions, the pH of the cultivating medium was maintained at aconstant pH of 7.3 by the automatic addition of 2 M sodium hydroxide.

For cell activation, the preceding culture in the flasks was used. Wheninoculum flasks reached late exponential growth phase (OD_(590nm)=0.5),an adapted volume of this culture was used to inoculate 1.8 L of freshmedium, which resulted in an initial OD of 0.4. The cells immediatelyentered into exponential growth for 3 hours with a doubling time of 42minutes (μ_(max)=1.00 h⁻¹). The 2-hour deceleration phase was followedby a stationary phase whereby the OD was 2.56. The same doubling timewas observed both in the flasks and in the fermentor. Although a slightimprovement was noted for the final OD, the biomass production yield(0.05 g/L·h) remained low. As observed in the fermentor, the pH wasconstantly maintained at 7.3. Furthermore, it was observed that glucosewas not the limiting source for GBS since the available glucose was 5.7g/L when the cells entered in the beginning of the deceleration phase.

B) Identification of Limiting Compounds

Based on the composition of yeast extract medium that is approximatelyknown, a comparison between the concentration of nutritional sourcesused in the complex medium process and the composition of the batchdefined medium was performed to determine an approximate ratio among thedifferent compounds required by GBS to reach a final OD around 15 and toidentify the limiting compounds for growth. The process involving thecomplex medium contained 17 g/L of yeast extract in the batch medium and19 g/L were added during the feed. Thus, 36 g/L were available forStreptococcus agalactiae.

The comparison between the composition of 36 g/L of yeast extract andinitial concentration of defined medium was performed on the mineral,glucose, vitamins and amino acids contents (see Table 3). For themineral sources, all compounds present in the current defined mediumwere sufficient to satisfy the GBS growth requirements, except potassiumwhich was 6.5 times more abundant in the complex medium. For the vitaminand amino acid sources, in all cases, the concentrations observed in theprocess using the defined medium were lower than the concentrations inthe process using the complex medium. The ratios of the vitamin or aminoacid concentration present in yeast extract compared to the batchconcentration in the defined medium were heterogeneous compared tonature of the molecules. However, the exact composition of the yeastextract is not well defined. Values used for the comparison are alsoaverages and the ratio of each component may differ significantlyaccording to the production process and processing of yeast extract.

TABLE 3 Comparison of composition complex medium and CDM process YEASTEXTRACT CDM Concen- Quantity for Final Chemical tration 36 g/L of yeastconcentration compounds (g/100 g) extract (mg) (mg · L⁻¹) Ratio Mineralcontents Calcium 120 43.2 — — Magnesium 200 72 194 0.371 Potassium 3.31188 193 6.15 Sodium <0.5 440 2430 0.181 Phosphorus 1.8 988 1370 0.721Iron 5 1800 2.14 0.841 Nitrogen — — 127 — Sulphate — — 1508 — Vitamincontents Biotin 0.25 0.09 0.01 9 Folic acid 3.1 1.116 0.1 11.2Niacinamide 68 24.5 2 12.2 Ca Panthotenate 30 10.8 0.8 13.5 Riboflavin11.9 4.28 0.4 10.7 Thiamine 3 1.08 0.4 2.7 Pyridoxamine 2.3 0.8289 0.81.03 VITAMINS: Average ratio yeast extract/CDM = 8.6 Free amino acidscontents L-Alanine 4.78 1721 200 8.605 L-Arginine 0.24 86.4 200 0.432L-Aspartic acid 2.49 896 100 8.96 L-Cysteine — — 200 — L-Glutamic acid6.01 2160 200 10.8 L-Glutamine — — 50 — Glycine 1.11 396 200 1.98L-Histidine 1.80 648 200 3.24 L-Isoleucine 2.64 950 100 9.504 L-Leucine4.34 1562 100 15.6 L-Lysine 3.08 1109 110 10.08 DL-Methionine 1.08 389100 3.89 L-Phenylalanine 2.72 979 100 9.79 L-Proline — — 200 — DL-Serine2.35 846 100 8.46 L-Threonine 2.02 728 100 7.28 L-Tryptophan — — 200 —L-Tyrosine 1.46 526 200 2.63 L-Valine 3.30 1188 100 11.88 AMINO ACIDS:Average ratio yeast extract/CDM = 7.5

Example 5 Extension of Chemically Defined Media to Fermentation

Fed batch fermentation typically starts as a batch mode, and after acertain biomass concentration or substrate consumption, the fermentor isfed with the limiting substrate solution. As such, the nutrients mediummust have a simple composition. The goal of this investigation was todevelop a batch medium that identifies the limiting compounds and thatdoes not affect the growth rate.

A) Development of Batch Medium for Fed Batch Process

In order to develop a defined fed batch medium, the limiting compoundswere added one by one, and their effects on growth were evaluated.

First, the concentration of each vitamin in Table 2 was increased by afactor of 10. The vitamins were observed to be very important for theGBS growth. The cells immediately entered an exponential phase whichlasted for 4 hours, reducing the doubling time from 42 minutes(μ_(max)=1.00h⁻¹) to 33 minutes (μ_(max)=1.26h⁻¹). After 3 hours, thecells had entered a deceleration phase for 2 hours before entering astationary phase after 5 hours of culture. The final OD was 3.70, whichwas 50% higher than the previous trial. When vitamins were added to thebatch medium, a positive effect was observed although they were not theonly limiting compounds. The exponential growth phase was finished after3 hours, but the glucose was still available at 6.3 g/L when the cellsentered the deceleration phase.

The same study was performed by adding both 10× vitamins and 10× aminoacids. For the fermentation involving 10× vitamins, the final OD washigher (OD_(590nm)=4.5). The exponential growth phase of the cellslasted for 4 hours. Glucose did not appear to be the limiting compoundssince 4 g/L was again present when the cells entered the decelerationphase.

A study was also performed by adding 10× of potassium to the initialmedium. In this case, a longer doubling time (t_(d)=49 minutes) anddecreased final OD (OD_(590nm)=2.9) were observed when the cells were inthe stationary growth phase. Increasing the initial potassiumconcentration by a factor of 10 had a negative effect on the growth.Thus, potassium needs to be added by feed or in smaller batches to avoidadding potassium to inhibitory levels.

According this study, the batch medium was composed of the same mineralcontents as the initial chemically defined medium, but the concentrationof vitamins and amino acids were increased by a factor of 10. Phosphateand carbon sources were added to the fed medium, but to determine theconcentration of each component, a new comparison to the process usingcomplex medium was necessary. Since 33 g/L of glucose were present inbatch medium and 55 g/L were added during the linear addition, in orderto add the same ratio of glucose and 10× of potassium, the fed mediumwas composed of: 275 g/L of glucose, 10.08 g/L of K₂HPO₄ and 14.8 g/L ofKH₂PO₄. 500 mL of this solution were added to 1.2 L of the batch medium.

B) Development of Fed Batch Process

The strategy for the fed batch fermentation was to feed the growthlimiting substrate at the same rate at which GBS consumed the substrate.

The nutrient feed rate influences fed batch fermentation by defining thegrowth rate of the microorganism and the effectiveness of the carboncycle for product formation and minimization of by-product.

C) Growth Factors Requirements of Group B Streptococci

An organism, whether it is an autotroph or a heterotroph, may requiresmall amounts of certain essential organic compounds for growth that theorganism is unable to synthesize from the available nutrients.

Growth factors are required in small amounts by cells because theyfulfill specific roles in biosynthesis. The need for a growth factorresults from either a blocked or a missing metabolic pathway in thecells. They are organized in three categories: (1) purines andpyrimidines required for synthesis of nucleic acids; (2) amino acidsrequired for the synthesis of proteins: and (3) vitamins needed ascoenzymes and functional groups of certain enzymes.

The purpose of this investigation was to identify the growth factorrequirements of the M781 strain of Streptococcus agalactiae to simplifythe cultivating medium by reducing the number of compounds and to ensurea cost-efficient production process.

D) Amino Acid Requirement of the M781 Strain of Streptococcus agalactiae

By eliminating the amino acids one by one from the medium, L-Alanine,L-Aspartic acid, L-Glutamine and L-Proline were found to be dispensable(See FIG. 20 and Table 4). These amino acids always resulted inturbidity values of greater than 80% in the control culture. However, inthe absence of any other amino acids, no growth occurred in thecultivating medium.

TABLE 4 Effect of omission of individual amino acids on growth of strainM781 of GBS in a CDM Percentage of control Amino acid DO final pH growthRequired All amino 1.985 4.70 acids L-Alanine 1.675 5.23 85 − L-Arginine0.053 7.10 3 + L-Aspartic 1.905 6.65 96 − acid L-Cystine 0.001 7.25 <1 +L-Glutamic 0.162 6.65 8 + acid L- 2.085 4.87 100 − Glutamine Glycine0.131 6.98 6 + L-Histidine 0.052 7.02 3 + L- 0.145 6.91 7 + IsoleucineL-Leucine 0 7.22 0 + L-Lysine 0.005 7.10 <1 + L- 0.045 7.06 2 +Methionine L- 0.008 7.20 <1 + Phenylalanine L-Proline 2.015 4.74 100 −L-Serine 0.401 6.44 20 + L- 0 7.22 0 + Threonine L- 0.133 6.96 7 +Tryptophan L-Tyrosine 0 7.20 0 + L-Valine 0 7.26 0 + +: On the absenceof the amino acid, M781 strain grew to 40% or less of the control −:Growth was at least 40% of the control culture.

Satisfactory growth in the cultivating medium was obtained when 15 aminoacids were present. Fermentation at a 2 L scale was performed to comparethe M781 growth with only the essential amino acids. The growth wasapproximately the same magnitude when 19 amino acids were present. Inboth cases, no lag phase was observed, but a doubling time reductionfrom 62 minutes (μ_(max)=0.672 h⁻¹) to 78 minutes (μ_(max)=0.533 h⁻¹)was observed when the four amino acids were removed from the chemicallydefined medium.

The impact on the OD and cps production by the M781 strain when these 4amino acids are omitted will be determined in the final process.

E) Vitamin Requirements of the M781 Strain of Streptococcus agalactiae

By eliminating the vitamins individually from the cultivating medium,calcium pantothenate and niacinamide were found to be indispensable(Table 5 and FIG. 21). In experiments where biotin, folic acid,pyridoxine, riboflavin and thiamine were omitted, the turbidity valueswere greater than 65%. When only pantothenate and niacinamide wereadded, the same final OD of the control was observed.

TABLE 5 Effect of omission of individual vitamins on growth of strainM781 of group B streptococci in a chemically defined medium Percentageof control Vitamins DO final pH growth Required All vitamins 1.63 4.60 —− present (Control culture) Effect of omission of individual vitamins ongrowth Biotin 1.71 4.50 100 − Calcium 0.020 7.02 <1 + panthotenate Folicacid 1.41 5.10 87 − Niacinamide 0.22 6.90 <1 + Pyridoxine 1.55 4.65 95 −Riboflavin 1.07 4.80 66 − Thiamine 1.37 4.55 84 − Omission of 5 vitaminsdetermined to not be required individually Calcium 1.58 4.53 96 −panthotenate & Niacinamide +: On the absence of the amino acid, M781strain grew to 40% or less of the control −: Growth was greater than 40%of the control culture.

Batch fermentation based on the original cultivating medium wasperformed using only calcium pantothenate and niacinamide. After 8 hoursof culture, the OD was 0.2. Thus, growth using only these two vitaminswas not optimal. A new study was performed in 500 mL Erlenmeyer flasksby adding individually the vitamins that were not required as determinedby the first shake flasks study to the medium already supplemented withcalcium pantothenate and niacinamide. In this study, as observed in thestudy with only calcium pantothenate and niacinamide, turbidity valuesof each flask after 18 hours of culture was equal to the controlculture.

Future experiments performed in the fermentor will enable theidentification of the necessary vitamins for the growth of the M781strain of Streptococcus agalactiae.

Example 6 Pilot-Scale Production

This Example confirms the teachings of the previous examples apply tothe manufacturing scale production of serotype specific capsularpolysaccharides of Streptococcal bacteria.

A) Culture of the Inoculum

i) Medium of Culture

The culture of the inoculum was performed in four 5 L-shake flaskssterilized by temperature (autoclave program n° 1 min./max., 40 min.,121° C., Table 6) containing 1 L of complex medium (17 g/L yeast extractDifco, 8 g/L Na₂HPO₄.2H₂O, 2 g/L NaH₂PO₄.H₂O, and 33 g/L monohydratedglucose, sterilized by 0.2 μm filtration with Nalgene filterwaredisposable systems, pH of 7.3±0.1 adjusted using 3 M NaOH), 10 mLsolution of vitamins (thiamine, riboflavin, pyridoxine HCl, andniacinamide, 0.05 g/L for each, diluted in 0.1 M NaOH, sterilized by 0.2μm filtration) and 5 mL of biotin solution (biotin 0.2 g/L, sterilizedby 0.2 μm filtration) added just before inoculation.

TABLE 6 Correlation between the content of the autoclave and thespecifications of sterilization Description of Number Sterilization thecontents of of the Organization Sterilization temperature the autoclaveprogram (b) time (min) (° C.) Liquid 7 2 or 4 30 121 Glassworks 4 1min., 1 max. 40 121 or 7 Dirty 8 3 130 124 Air Filter (a) 2 NA 40 121Liquid 6 6 50 121 Antifoam 9 8 60 121

ii) Inoculation of the Flasks and Conditions of Culture

Each flask was inoculated with 2.75±0.25 mL of working seedsextemporaneously defrosted from the −70° C. freezer. The culture wasmaintained at 35±1° C. with agitation 200±10 rpm in the incubator(IN-L0641) during 4±1 h. After this time, the biomass concentration wasevaluated by measuring the OD at 590 nm and performing a Gram stain. Ifthe value of OD_(590nm) reached 1.2-0.6, and if the Gram stain conformed(only Gram positive cocci), the contents of the four flasks were pooledinto a 5 L heat-sterilized (autoclave program n° 1 min./max.) bottlewith connections to incubation line of the 300 L B. Braun Biotech/Chemapfermentor (ID VS-L0530).

iii) Key Variables of the Inoculum Preparation

The key variables of the inoculum preparation are described in Table 7and Table 8.

TABLE 7 Controlled variables during the inoculum preparation ControlledVariables Target Range Initial pH of the medium 7.3 ± 0.1 Volume ofworking seed 2.5-3.0 ml/flask Temperature of incubation 35 ± 1° C.Agitation speed 200 ± 10 rpm

TABLE 8 Monitored variables during the inoculum preparation MonitoredVariables Target Range Final OD_(590 nm) 0.6-1.8 GRAM Only Gram positivecocci Culture purity No contaminant Time of incubation 3-5 hours

iv) Sterilization and Cleaning of Equipments

After use, the flasks and the 5 L bottle were heat sterilized (autoclaveprogram n° 8, dirty cycle, see Table 6 for specifications) and cleaned.

B) Culture in the 300 L Fermentor

i) Preparation of the Medium and Equipment

The mechanical piping and gas filters of the empty 300 L fermentor weresterilized (program SEAL2 and EXFC2). The probes are then checked andcalibrated. The pH probe was calibrated using two buffer solutions withvalues of pH 7 and 10. The correct application of the oxygen prove wasverified by putting the probe in water with a gas-flow of nitrogen forthe 0% point and air for the 100% point. Its calibration was performedinside the fermentor.

The basic medium (120 L, 2 g/L Na₂HPO₄.2H₂O, 1 mL for 120 L antifoam“PPG 2500”) was formulated and sterilized in the 300 L fermentor(program FVES 2). During the sterilization, the 0% value of the oxygenprove was checked and reinitialized if necessary. After the coolingphases of the sterilization, the temperature of medium reached 36° C.,and the basic medium was completed with 17 L of yeast extract 150 g/L, 9L of glucose monohydrated 550 g/L, 2 L of a solution of vitamins(Thiamine, Riboflavin, Pyridoxine HCl, and Niacinamide, 0.05 g/L foreach, diluted in 0.1 M NaOH) all sterilized by 0.2 μm filtration. The100% value of the oxygen prove was then calibrated after oxygenation ofthe medium. After 4 L of the inoculum were added, the final volume was150 L at the beginning of the fermentation and the final concentrationof yeast extract was 17 g/L and glucose was 33 g/L. These additions wereperformed on sterilizable lines with a peristaltic pump at maximalvelocity (400 rpm) that corresponded to a flow of 550 mL/min.

ii) Fermentation Process and In-Process Controls

Before the inoculation, a biotin solution (IL, 0.2 g/L biotin,sterilized by 0.2 μm filtration) was added. The 300 L fermentor was theninoculated using the 5 L bottle containing the content of the 4 flasksof inoculum.

The value of the following parameters were then checked, adjusted ifnecessary and automatically controlled during the process:

-   -   the temperature of the culture was controlled at 36±P° C.,    -   the overpressure inside the fermentor was set at 0.2 bar,    -   the pH was set at 7.3±0.1 and adjusted using 4 M NaOH. There was        no pH correction using an acidic solution because the pH value        naturally decreased due to fermentation.    -   the initial stir was set at 50 rpm and the initial airflow was        set at 20 L/min,    -   the level of foam in the fermentor was visually monitored and        adjusted using antifoam PPG 2500 if necessary,    -   the dissolved oxygen tension (DOT) was set at 30% regulated in        cascade by:        -   the stir (range of values between 50 and 350 rpm)        -   the airflow (range of values between 20 and 100 L/min)        -   the oxygen flow (range of values between 0 and 100 L/min)

Samples were taken during the batch phase of the fermentation, two hoursafter inoculation, and the OD_(590nm), was measured. Samples were takenevery 15 minutes until the OD_(590nm) reached 3. At that target OD, thefirst exponential fed batch addition was initiated using 3.6 L of ayeast extract solution (150 g/L), maintaining the population doublingtime at 300 minutes.

Approximately 45 minutes after the first addition, the OD_(590nm) wasmeasured. Samples were taken every 15 minutes until the OD_(590nm)reached 5. At that target OD, a second exponential fed batch additionwas initiated using yeast extract solution (150 g/L), maintaining thedoubling time at 50 minutes.

At the end of this second exponential fed batch addition, a pH-stat fedbatch addition was performed. A monohydrated glucose solution (550 g/L)was added when the pH value exceeded 7.18. During this addition, asample was taken every hour to measure the OD_(590nm).

The fermentation finished approximately 3 hours after the last addition.The automatic controls of the parameters were then stopped. The stir wasregulated at 100 rpm and the temperature at 30° C.

iii) Key Variables of the Fermentation Process

The key variables of the fermentation process are described in Table 9and Table 10.

TABLE 9 Controlled variables during the fermentation ControlledVariables Target Range pH of the medium 7.3 ± 0.1 DOT setpoint 30%Temperature 36 ± 1° C. Overpressure 0.2 bar

TABLE 10 Monitored variables during the fermentation Monitored VariablesTarget Range Sterility check before inoculation No contaminantOD_(590nm) for each sample — Culture purity at the end of thefermentation Lack of contaminant GRAM test Only Gram positive cocciFermentation time —

iv) Sanitization, Sterilization and Cleaning of the Equipments

Once the biomass was removed from the 300 L fermentor, the sanitizationwas initiated by adding 200 L of ROW into the fermentor. 3 M NaOH wasthen added into the fermentor until the pH reached 11. The temperaturewas maintained at 80° C. for 30 minutes. After cooling to ambienttemperature, the content of the fermentor was discarded into the wastetank located at the lower floor.

The sterilization was then performed by adding 200 L of ROW andactivating the program (FVES 2) according to the Standard OperatingProcedures (SOP). After the cooling phases of the sterilization, the pHand oxygen probe were removed from the fermentor and respectivelystocked in a 3 M KCl solution and ROW.

The fermentor was finally washed using 200 L of 1 M NaOH, and stirred at100 rpm for at least 30 minutes. This 200 L of NaOH were emptied intothe killer tank after the washing and other 100 L of NaOH were placed inthe fermentor via a spray ball so as to clean the upper part of thevessel. These 100 L were recirculated using a lobe pump for a minimum of30 minutes. After this cleaning step, the fermentor was washed with ROWuntil the pH decreased to a range between 5 and 7.

C) Centrifugation of the Biomass

i) Equipment Preparation

A tank containing physiological water (˜100 L, 9 g/L NaCl, sterilized by0.2 μm filtration) was connected to the transfer line that joined the300 L fermentor to the Alfa-Laval centrifuge (ID CT-L0526). This waterwas used during the centrifugation to wash the biomass pellet. Thetransfer line was then heat sterilized like the separator and thecollector tank of biomass. The preparation of equipments was performedbefore the end of the fermentation in order to begin the centrifugationas soon as possible after the end of the fermentation.

ii) Continuous Flow Centrifugation Process

The continuous flow centrifugation process was composed of the followingcycle:

-   -   7 minutes of biomass centrifugation at a flow of 100 L/h,        manually adjusted. The biomass was transferred from the        fermentor to the separator through the transfer line by an        excess pressure of 0.6 bar in the fermentor.    -   3 minutes of washing with physiologic water at a flow of 100        L/h, manually adjusted,    -   discharge of the pellet.

This cycle was usually repeated until the entire biomass was processed.The supernatant was not collected, but instead was fed to the wastetank. The pellet was collected in the tank (VS-L0536) during theprocess, and then transferred by an excess pressure of 0.3 bar through asilicone connection to a 100 L disposable sterile bag for chemicaltreatments.

iii) Key Variables of the Centrifugation

The key variables of the fermentation process are described in Table 11.

TABLE 11 Controlled variables during the centrifugation ControlledVariables Target values Pressure in the fermentor 0.6 ± 0.1 bar Flow 100L/h Temperature of biomass 30 ± 1° C. Discharge time 10 (7 + 3) min (a)Temperature of the pellet 30 ± 1° C. Pressure of the supernatant 3.0 ±0.3 bar NOTE: 7 minutes of biomass centrifugation + 3 minutes of washingwith physiological solution.

iv) Sterilization and Cleaning of the Equipments

After the centrifugation and transfer of the pellet to the disposablebag, the transfer line, the separator and the collector tank of biomasswere heat sterilized and cleaned. The transfer line and separator werecleaned using 100 L of 1 M NaOH at ambient temperature in the fermentor,and then transferred through the transfer line to the separator at aflow of 100 L/h. The collector tank was cleaned by circulating 20 L of 1M NaOH for 30 minutes in the tank through a spay ball so as to clean theupper part of the tank. After this cleaning step, the tank was washedwith ROW until pH decreased to a range between 5 and 7.

D) Chemical Treatments of the Cellular Pellet

i) Inactivation of the Cellular Pellet

The chemical treatment of the cellular pellet inactivated the bacteriaand enabled the release of cps from the bacteria. The treatment involvedthe addition of a 4 M NaOH solution (through a tube with a peristalticpump) to the pellet to obtain a theoretical concentration of 0.8 M NaOH.The weight of the 4 M NaOH added was obtained by dividing the biomassweight by four since IL weighed 1 kg. This step was performed in a 100 Ldisposable bag with an integrated stirrer system and disposed in theLevtech Sartorius System (ID AG-10645, thermostated balance andstirrer). The temperature was regulated to maintain the pellet at 37° C.and then stirred at 180 rpm for a specified period of time. 12 h wereenough to inactivate the microorganisms. 36 h were suitable forreleasing the cps from the bacterial capsule. In other experiments, 1 hwas found to be enough to inactivate the microorganisms, while 24h weresuitable for releasing the cps from the bacterial capsule. Accordingly,a total time of 36h or 24h is suitable for this step.

ii) Key Variables of the Inactivation

The key variables of the fermentation process are described in Table 12.

TABLE 12 Controlled variables during the inactivation and release of theCPS Controlled Variables Target values Temperature of inactivation 37 ±0.1° C. Agitation 180 ± 10 rpm Time 36 ore

iii) Neutralization and Precipitation

Using silicone tubes with a peristaltic pump, a buffer solution of TRIS1 M is added to obtain a final concentration equal to 0.1 M. The weightof TRIS to add is calculated by dividing the weight of the inactivatedbiomass by 9. The importance of this addition was to avoid pH variationin the pellet during the neutralization. Thus, the pH was controlledusing a pH probe disposed in the disposable bag. 6 M HCl was added toobtain a final pH value of 7.5-8.5.

2 M CaCl₂ and 96% ethanol solutions were added to precipitate proteinsand nucleic acids in the pellet. The final CaCl₂ concentration was 0.05M and ethanol was 30%. The weight of the 2 M CaCl₂ added was obtained bydividing the weight of the neutralized biomass by 19, and the weight ofethanol 96% was obtained dividing the weight of the neutralized biomasswith CaCl₂ by 3.1.

E) Microfiltration and Dialyze of the Treated Pellet

The biomass, chemically treated with CaCl₂ and 30% ethanol, underwent amicrofiltration to recover the polysaccharides released in thesupernatant and to eliminate the cellular residues, as well as theprotein and nucleic acid precipitates.

i) Equipment Preparation

The microfiltration and dialysis were performed using a SartoriusSartocon II plus holder with a disposable housing, and 4 Hydrosartcassettes 0.22 μm, 0.6 m² which represented a total surface area of 2.4m². The system was tightened using a torque wrench of 90 Nm.

The cassettes were sanitized using 20 L of 1 M NaOH, and sterilized by0.2 μm filtration using a lobe pump to assure and regulate the pressurein the system. The retentate and permeate were then recirculated for 30minutes in the following conditions:

-   -   Inlet pressure: 2.0±0.2 bar    -   Permeate valve closed for 5 minutes and then widely opened.

Distilled water was used to was the system until the pH reached 5-7, atwhich time the system was washed with 20 L of physiologic water (0.9 g/LNaCl, sterilized using a 0.2 μm filtration) to obtain a pH of 5-7 in thefollowing conditions:

-   -   Inlet pressure: P_(in)=2.0±0.2 bar    -   Permeate pressure: P_(perm)=0 bar (open valve)    -   Retentate valve closed.

Prior to the microfiltration, the cassettes were conditioned with thedialysis buffer solution (34.77 g/L NaCl, 4.49 g/L TRIS, 10.93 g/LCaCl₂, pH adjusted to 7.8±0.1 using 6 M HCl, WFI qsp 74.4% of finalvolume. 96% ethanol until final volume, sterilized by 0.2 μmfiltration).

ii) Microfiltration and Dialyze

The exit tube of the disposable bag containing the treated biomass wasconnected to the inlet of the microfiltration housing. The retentateexit of the housing was connected to the disposable bag that containedthe treated biomass to recirculate the processed biomass. The permeateexit was connected to a 200 L disposable sterile bag to collect thepermeate.

The permeate valve was initially closed to let the pellet circulate inthe microfiltration system. This valve was then opened, and the velocityof the lobe pump was controlled to obtain the following conditions:

-   -   Inlet pressure: P_(in)=2.0±0.2 bar    -   Permeate pressure: P_(perm)=0.6±0.1 bar

The biomass was concentrated 10 times, and the retentate was dialyzedagainst 3 volumes of buffer. To accurately determine the circulation inthe microfiltration system, the weight of the retentate must not be lessthan 10 kg. As such, the retentate was concentrated until the biomassweighed 10±0.5 kg. The dialysis was then performed in successive steps.The weight of buffer solution used was calculated from the weight of theCaCl₂-ethanol mix divided by the theoretical concentration factor 10 andmultiplied by the desired number of dialysis cycles.

iii) Sterilization by Filtration of the Permeate

The permeate was sterilized by filtration using a 2000 cm² Sartobran P0.22 μm filter at the exit of the microfiltration system beforecollection into the 200 L disposable bag. The final product was stockedat ambient temperature before release into the purification department.

iv) Key Variables of the Microfiltration and Dialyze

The key variables of the microfiltration and dialyze are described inTable 13.

TABLE 13 Controlled variables during the microfiltration and dialyzeControlled Variables Target values P_(in) 2 ± 0.2° C. P_(perm) 0.6 ± 0.1bar Retentate temperature ≦20° C.

The monitored variables are the permeate flow and the quantity ofpolysaccharides.

v) Equipment Cleaning

After the microfiltration, the inlet was connected to a tank containing100 L of physiological water to wash the disposable cassettes andhousing. Then, 20 L of 1 M NaOH were used to sanitize the system, andthe permeate and retentate were connected to the inlet for recirculationfor 30 minutes while maintaining the following conditions:

-   -   Inlet pressure: P_(in)=2.0±0.2 bar    -   Permeate valve closed for 5 minutes and then widely opened.

The system and piping were then emptied, and washed with distilled wateruntil the values of pH of the permeate and retentate ranged between 5and 7. At that target pH, the system was disassembled.

An integrity test of the Sartobran P filter used to sterilized thepermeate during the microfiltration and dialyze was performed before therelease of the batch to the purification department.

F) Description of the Fermentation Profiles

The fermentation profiles of the pilot-scale experiments correspondingto the 3 GBS strains. M781 (serotype III), H36b (serotype Ib) and O90(serotype Ia), were analyzed and compared with a control fermentationperformed at laboratory-scale in a 30 L fermentor (B. Braun BiotechBiostat) using the H36b strain an identical process.

The OD_(590nm) profiles of the 3 pilot-scale fermentations were verysimilar to each other, as well as to the control fermentation (see FIG.22). The general profile of the microorganism's growth can be describedin the following way: The batch phase lasted approximately 2.5 hours,and resulted in an OD_(590nm) equal to 3. The first exponential fedaddition of yeast extract solution (F1, 150 g/L) lasted approximately 45minutes, and resulted in an OD_(590nm) equal to 5. The secondexponential fed addition of yeast extract solution (F2, 150 g/L) lastedapproximately 45 minutes, and resulted in an OD_(590nm) of approximately10. The third pH-stat fed addition of monohydrated glucose (F3, 550 g/L)lasted approximately 3 hours.

G) Evaluation of the Growth Rates and Population Doubling Times

The growth rates (μ) and population doubling time (t_(d)) were evaluatedduring the 3 pre-test runs and the control fermentation. The values werereported in FIG. 22, as well as Table 14.

TABLE 14 Growth rate and population doubling time during the firstseries of the pre-trial runs. Phase M781 H36b O90 μ_(F1) (h⁻¹) F1 0.590.96 0.54 td_(F1) (min) 71 43 88 μ_(F2) (h⁻¹) F2 1.01 0.89 0.65 td_(F2)(min) 41 47 64 μ_(F3) (h⁻¹) F3 0.20 0.23 0.23 td_(F3) (min) 203 182 182

The population doubling times during the first exponential fed additionof the pre-test runs, ranged between 43 and 77 minutes, which was atleast as good as the reference fermentation. However, the desiredpopulation doubling time was 30 minutes. During the second exponentialfed addition of the pre-test runs, the population doubling times rangedbetween 41 and 64 minutes, which was almost equal to the referencefermentation (55 minutes) and very near of the theoretical populationdoubling time (50 minutes).

H) Evaluation of the Production of Capsular Polysaccharides

The concentration of cps was evaluated at the end of fermentation usinga colorimetric method based on the determination of the concentration ofsialic acid compound of the cps. Based on this result, the cps quantityproduced during the culture was calculated by multiplying the cpsconcentration by the final volume inside the fermentor (20 L for thereference fermentation, 215 L for the pilot-scale fermentor). Thevolumetric and specific productivity were also calculated as set out inthe Fermentation Related Analytical Methods below. The values werereported in Table 15.

TABLE 15 Production and productivity of capsular polysaccharides M781H36b O90 Final OD_(590 nm) 18.15 25.1 14.0 Final concentration of dried7.93 10.97 6.12 biomass (g/L) Final concentration of cps (g/L) 0.38 0.310.42 Quantity of produced cps (g) 82 67 90 Ratio cps/biomass (%) 4.8 2.86.8

I) Simplification of the Initial Process

The fermentation process was simplified in two ways to avoid potentialvariations in the scale-up and to decrease the risk of contamination inthe fermentor.

First, the vitamin solutions containing thiamine, riboflavin, pyridoxineHCl, and niacinamide (each 0.05 g/L diluted in 0.1 M NaOH and sterilizedby 0.2 μm filtration) were removed from the medium of the inoculum andthe fermentor. Indeed, the laboratory results had shown that theaddition of these vitamins were not necessary for the growth of GBS andhad a negligible impact on the cps production.

Second, the parameters of the fed phases during the fermentation weremodified. The two exponential fed phases of yeast extract addition werereplaced by two instantaneous additions, and the pH-stat fed phase ofglucose addition was replaced by a linear addition. The firstinstantaneous addition (F1) comprised of adding a 3.6 L solution ofyeast extract, 150 g/L using a peristaltic pump at a flow of 550mL·min⁻¹ when the OD_(590nm) was in the range between 2.5 and 3. Thesecond instantaneous addition (F2) comprised of adding a 13.4 L solutionof yeast extract, 150 g/L using a peristaltic pump at a flow of 550mL·min⁻¹ when the OD_(590nm) was in the range between 4.5 and 5. Thethird linear addition (F3) comprised of adding a 17 L glucose solutionusing a peristaltic pump at a flow of 95 mL·min⁻¹ when the OD_(590nm)was in the range between 10 and 12.

J) Description of the Fermentation Profiles and Comparison with thePreceding Runs

The fermentation profiles of this series of pre-test runs were analyzedand compared with the first series to ensure that the simplified processdid not have any impact at the pilot-scale.

The OD_(590nm) profiles of the 3 pilot-scale fermentations were verysimilar to each other, as well as to the general profile observed in thefirst series of pre-test runs (see FIG. 23). This similarity between theprofiles highlights that the modifications of the process have no impacton the growth of the microorganisms as demonstrated at laboratory-scale.

K) Evaluation of the Growth Rates, Population Doubling Times andProduction of Capsular Polysaccharides

The growth rates and population doubling times of this series (Table 16)were also very similar to the first series of pre-test runs and nosignificant variations were observed.

TABLE 16 Growth rate and population doubling time during the secondseries of pre-trial runs Phase M781 H36b O90 μ_(F1) (h⁻¹) F1 0.80 0.660.67 td_(F1) (min) 52 63 63 μ_(F2) (h⁻¹) F2 0.92 0.76 0.92 td_(F2) (min)45 55 45 μ_(F3) (h⁻¹) F3 0.18 0.31 0.19 td_(F3) (min) 232 133 219

The final cps concentration of these pre-test runs were in accordancewith the previous pre-test runs. This suggests that there was nosignificant difference that resulted from the modifications at eitherthe laboratory-scale or pilot-scale (see Table 17).

TABLE 17 Production and productivity of capsular polysaccharides M781H36b O90 CJB111 Final OD_(590 nm) 18.25 20.7 17.5 25.5 Finalconcentration of dried 7.98 9.05 7.65 10.5 biomass (g/L) Finalconcentration of cps (g/L) 0.26 0.30 0.40 0.37 Quantity of produced cps(g) 56 65 86 86 Ratio cps/biomass (%) 3.2 3.3 5.2 3.4%

L) Critical Steps of the Process and Definition of the Sampling Plan forthe Process Verification

During these two series of pilot-scale test runs, important in-processcontrols of the critical processing steps had been defined with theiracceptance criteria and the associated sampling plan. The firstin-process control was the OD_(590nm) of the flask before inoculationthat is preferably between 0.6 and 1.8 to avoid a potential lag phase atthe beginning of the cultivation in fermentor as was observed at thelaboratory-scale. The following in-process controls were relevant to thepurity of the culture: Gram stains of the flasks medium were performed,as well as spreading on plates of the pooled bottle and the medium ofthe fermentor, to ensure the environment was free of contaminants.Another Gram staining and spreading on plates of the medium inside thefermentor was performed at the end of the culture to verify that therewas no contamination during the process. The inactivation of the pelletwas verified by spreading the pellet in 0.8 M NaOH on the plates afterthe 36 hours of inactivation. The other in-process controls described inTable 18 were used to calculate the cps purification yields. A profileof the parameter variations (P_(O2), air flow, O₂ flow, stir, pH,temperature) was developed, and was a good indicator of thereproducibility of the fermentation process.

i) Description of the General Fermentation Profiles and Comparison withthe Previous Runs

The OD_(590nm) profiles of this test run were very similar to eachother, as well as to the general profile observed in the earlier testruns in this Example (see FIG. 24).

The growth rates and population doubling times of this series (Table 18)were comparable to the pre-test runs. More specifically, the growthrates of the addition phases of the 3 test runs were between the minimaland maximal values of the growth rates previously reported. However, thegrowth rates of the F3 phase during the culture of M781 and of the F2phase during the culture of O90 were slightly below the minimum valuepreviously reported (respectively 0.15<0.18, and 0.62<0.65) but withoutany incidence on the final values of OD_(590nm) that were between theextreme values of OD_(590nm) obtained during the earlier test runs (14and 25.1 respectively for the first pre test run of the strain O90 andH36b).

TABLE 18 Growth rate and population doubling time during the series oftest runs Phase M781 H36b O90 CJB111 μ_(F1) (h⁻¹) F1 0.82 0.85 0.63 0.72td_(F1) (min) 51 49 60 58 μ_(F2) (h⁻¹) F2 0.92 0.96 0.62 1.16 td_(F2)(min) 45 43 67 36 μ_(F3) (h⁻¹) F3 0.15 0.21 0.17 0.27 td_(F3) (min) 281201 242 158

The final concentration and quantity of cps of the test runs were higherthan the pre-test runs when the results each of the 3 strains werecompared (see Table 19). The value for the H36b stain was between thevalues previously obtained for the other strains (0.35 L⁻¹ between 0.26and 0.42 respectively obtained for the first pre-test run of O90 and thesecond one of M781). The values obtained for the strains M781 and O90were higher than expected. As such, the cps to biomass ratios for thesestains exceeded 10%, which implied that the purification would befacilitated.

TABLE 19 Production and productivity of capsular polysaccharides M781H36b O90 CJB111 Final OD_(590 nm) 17.2 20.9 16.5 26.55 Finalconcentration of dried 7.52 9.12 7.21 11.7 biomass (g/L) Finalconcentration of cps (g/L) 1.01 0.35 0.82 0.42 Quantity of produced cps(g) 216 75 177 90.3 Ratio cps/biomass (%) 13.4 3.8 11.4 3.6

Analysis of the Critical In-Process Controls

The first in-process control was the OD_(590nm) of the flask beforeinoculation, and ranged between 0.80 for strain O90 and 1.50 for strainM781. No lag phase at the beginning of the culture in fermentor wasobserved. The purity analysis of the flasks culture were confirmed byGram stain which revealed only Gram positive cocci, and the pooledbottle as well as medium from the fermentor at the end of the culturesimilarly only revealed Gram positive cocci. The inactivation of thepellet in 0.8 M NaOH was spread on the plates after the 36 hours ofinactivation.

The profiles of the variations of parameters (P_(O2), air flow, O₂ flow,stir, pH, temperature) during the 4 test runs were comparable to thegeneral profile that was reported in the pre-test runs.

TABLE 20 Results from Pilot Runs (Polysaccharide Purification) Serotype:Ia Ib III V Parameter Unit Limit Test Run Test Run Test Run Test RunFinal product weight g N/A 25.9 21.1 28.9 15.5 TGA (dry weight) %, w/wN/A 93.6 90.9 91.9 90.2 Saccharide titer μg/mg* >850 1030 989 959 1025Proteins μg/mg* <10 <5 <5.5 <5 <5 Nucleic acids μg/mg* <10 <0.0006<0.002 0.007 <0.00006 Group Polysaccharide μg/mg* <10 <2 <1.8 <2 <2 Freesialic acid %, m/m <1 <0.8 <0.9 <0.8 <0.15 Structural conformity N/Aconform conform conform conform conform N-acetylation degree % >80 92 9290 103 Kd N/A N/A 0.481 0.330 0.463 0.401 Endotoxins/saccharide Ul/μg <1<0.0005 <0.001 0.0007 0.0003 Antifoam μg/mg <10 <10 <10 <10 nd *μg/mg ofdry weight

TABLE 21 Results from Pilot Runs (Activation/Conjugation) Serotype: IaIb III V Unit Limit Test Run Test Run Test Run Test Run WorkedPolysacchar. g N/A 6.64 7.19 7.0 5.08 Worked Product*** L N/A 51 68 4966 Final Product Kg N/A 3.50 3.89 4.60 1.69 Saccharide concentr. μg/mLN/A 1133 999 489 781 Protein concentration μg/mL N/A 427 594 384 678Glycosylation Degree N/A (1) 2.7 1.7 1.3 1.2 Free Protein %, w/w <5 <5<3 <5 <2 Free Saccharide %, w/w <25  16.2 8.9 19.6 <1 Free Sialic acid%, m/m <1 <0.3 <0.3 <0.7 N/A pH N/A 6.9-7.5 7.2 7.1 7.2 7.2Identity/Conformity* N/A positive positive positive positive positiveNaCNBH3 ppm <2 <2 <2 <2 <2 Kd N/A N/A 0.212 0.220 0.351 0.40Endotoxins/saccharide Ul/mg** N/A 1 0.3 0.1 0.24 Total saccharide g N/A4.0 4.4 2.3 1.3 (1): Ia and Ib: 1.0-3.5; III: 0.5-2.5; V: 0.5-3.0 *byNMR; **mg of saccharide; ***as equivalent fermentation volume

TABLE 22 Forecast Yields for 1000 L upscale (based on Pilot Processes)Purified Glycoconjugate Number of 20 μg Polysaccharide Saccharide dosesForecast Polysaccharide expected from expected from a expected fromNumber of expected form a a 1000 L culture 1000 L culture a 1000 L batchdoses 1000 L batch batch batch (according to expected from (according to(according to (according to Pilot optimized Pilot Processes) PilotProcesses) Pilot Processes) Processes) processes Serotype (g) (g) (g)(Million) (Million) Ia 370 140 84 4.2 >8 Ib 370 110 62 3.1 >6 III 370125 38 1.9 >4 V 370 75 20 1.0 >3

Fermentation Related Analytical Methods

Determination of Biomass.

During fermentation, biomass content is monitored by measurement of theoptical density of the culture at a wavelength of 590 nm. Dilutions ofthe sample have to be prepared in order to read a value of absorbancewithin the interval 0.300-0.600. Wet weight of harvest is determinedafter centrifugation for 25 min at 16000×g.

Determination of Capsular Polysaccharide Content.

The serotype-specific capsular polysaccharide of GBS is made of arepeating unit of the following saccharides: NANA: N-acetyl-neuraminicacid or sialic acid; GLUC glucose; GAL: galactose and NAGA:N-acetyl-glucosamine. Sialic acid content can be determined using thechemical method set-up by Svennerholm (Svennerholm L., (1957) Biochem.Biophys. ACTA 24:604-611). The composition of the repeating unit differswith serotype, so a different correction factor has to be applied foreach serotype.

repeating unit FW Ratio Correction Sugar FW serotype gal:glu:naga:sialCP NANA/CP Factor Ia 2:1:1:1 980 0.315 3.17 NANA 309 Ib 2:1:1:1 9800.315 3.17 GLUC 180 II 3:2:1:1 1304 0.237 4.22 GAL 180 III 1:2:1:1 9800.315 3.17 NAGA 221 V 2:3:1:1 1304 0.237 4.22

Sample Preparation.

A quantity of 10 OD·mL is centrifuged (16000×g, 5 min, 4° C.).

[standardise]

Wash the pellet with 1 mL of PBS and centrifuge [wash] (16000xg, 5 min,4° C.) To the pellet is added 500 mcL of NaOH (2N, 65° C., 1 h)[hydrolise] After 1 hour, neutralize with 500 mcL, HCl (2N, 4° C.)[neutralise] Cell debris are removed by centrifugation [purify](16000xg, 30 min, 4° C.). Supernatant is sterilized by filtration (0.22micron) [sterilise] 100 mL are diluted with 900 mL of H₂O [dilute]

Standard Curve Preparation.

A culture of strain COHI-13 (unencapsulated) is prepared in the same wayas the samples. The dilution step is characterised with the addition ofa known quantities of sialic acid stock solution to obtain finalconcentrations of 1, 5, 10, 15, 20 and 30 mg/mL. (100 mL supernatant+×mLsialic acid SS+900-×mL H₂O)

Chemical Reaction.

Starting Materials for the reagent: A=Resorcinol (2%, H₂O); B=CuSO₄.5H₂O(0.1 M, H₂O). Fresh reagent is mixed as follows: 10 mL A+0.25 mL B+H₂O(V_(fin)=20 mL)->+HCl (37%) (V_(fin)=100 mL). Reagent once mixed isstable for 1 week at 4° C. Add 1 mL of reagent to 1 mL of dilutedsample, incubate for 40 min at 90° C., read absorbance at 564 nm.

Quantification.

Determine quantity of NANA in sample using standard curve.

Apply correction factor of the serotype. [specific CP content (mg/LOD)]Multiply with OD of culture. [volumetric CP content (mcg/mL or mg/L)]Multiply with volume of harvest [total CP produced (mg)]

Example 6 Purification

This example shows an exemplary purification protocol which providesmuch higher levels of purity than have previously been possible forcapsular polysaccharides

Isolation and Purification of GBS Type Ia, III and V Polysaccharides

Native GBS Type V polysaccharide were extracted and purified frombacteria using the process steps:

Bacterial Fermentation:

GBS Type V strain (e.g., CJB111) was grown complex medium. Any method ofculture may be used, though fermentative culture as disclosed herein ispreferred.

Inactivation of Fermentation Biomass and Polysaccharide Extraction (BaseTreatment):

If necessary, the biomass may be heated to bring it to room temperature.Sodium hydroxide (4 M) was added to the recovered biomass to a finalconcentration of 0.8 M and mixed to homogeneity. The suspension wassubsequently incubated at 37° C. for 36 hours with mixing.

Neutralization of Biomass:

After extraction with base treatment, TRIS-base 1 M (121.14 g/mol) wasadded to a final concentration of 50 mM (52.6 mL per 1 L of basemixture) and the suspension was mixed to homogeneity. The pH of themixture was adjusted to 7.8 with HCl (6 M) (1:1 dilution of theconcentrated acid).

Alcohol Precipitation:

2 M CaCl₂ was added to a final concentration of 0.1 M (52.6 mL per 1 Lof neutralized mixture) and the suspension was mixed to homogeneity.Ethanol (96% (v/v)) was added to a final concentration of 30% (v/v)ethanol (428 mL per 1 L) and the suspension was mixed to homogeneity.

Tangential Microfiltration:

The supernatant from the alcohol precipitation was recovered by atangential microfiltration on a 0.2 μm cellulose membrane (SartoriusSartocon Hydrosart 0.1 m2) against a dialysis buffer comprising: NaCl(0.5 M)+CaCl₂ (0.1 M)+Ethanol 30% (v/v) buffered at pH 7.8. Ten dialysisvolumes were used for the microfiltration. The permeate was filteredusing a 0.45/0.2 μm filter to sterilize the permeate (SartoriusSartobran filter). Note: as an alternative, the retentate can beclarified by centrifugation (retaining the supernatant fluid) and storedat 2-8° C.

Tangential Diafiltration 30 kDa:

To eliminate particulate matter formed during storage, the material wasfiltered with a 0.45/0.2 μm filter (Sartobran filter). The material wasdia-filtered on 30 kDa cellulose membrane (Sartorius Sartocon Hydrosart0.1 m²) against 25 volumes of TRIS 50 mM+NaCl 0.5 M buffered at pH 8.8and then against 10 volumes of Na₂CO₂ 0.3 M+NaCl 0.3 M buffered at pH8.8. Pressure setting: ΔP[P_(in)−P_(out)]<0.7 bar,TMP[(P_(in)+P_(out))/2]>1.0 (e.g., P_(in)=2 bar, P_(out)=1 bar). Theretentate was filter sterilized using a 0.45/0.2 μm filter (SartoriusSartobran filter). The material was then stored at 2-8° C. until needed(max 15 days).

Depth Filtration:

A depth filtration on CUNO BioCap 2000 1300 cm² capsule (or CUNOZ-Carbon R52SP filter for smaller scale preparation) was applied toremove residual protein contaminants. The number of capsules or filtersused was defined on the base of the ratio: 0.5 cm² per mg of residualproteins.

Example with CUNO Capsules:

Using a peristaltic pump, the capsule was washed with >9.0 L of Na₂CO₃300 mM+NaCl 0.3 M buffered at pH 8.8 at flow rate of 350±50 mL/min. Ifthe volume of the material was less than 1.6 L, the suspension wasdiluted to the right volume with Na₂CO₃ 0.3 M+NaCl 0.3 M buffered at pH8.8. The material was filtered, and the filter was subsequently washedwith 2.5 L of Na₂CO₃ 0.3 M+NaCl 0.3 M buffered at pH 8.8. The materialobtained from the different capsules was combined. The collectedmaterial was filtered on new capsules (⅕ of the previous number) andwashed with 2.5 L of Na₂CO₃ 0.3 M+NaCl 0.3 M buffered at pH 8.8. Thematerial was filter sterilized using a 0.45/0.2 μm filter (SartoriusSartobran filter). The material was stored at 2-8° C. until needed (max15 days).

Re-N-Acetylation of Polysaccharide:

The material was diluted to 2 mg of polysaccharide/mL (estimated byresorcinol sialic acid assay) with Na₂CO; (0.3) M+NaCl (0.3 M) bufferedat pH 8.8. Stock solution of acetic anhydride was prepared at thefollowing proportions: 8.3 mL of acetic anhydride+8.3 mL of Ethanol96%+983.4 mL of water. Fresh acetic anhydride stock solution was addedto the polysaccharide solution diluted to 2 mg/mL to a ratio of >22:1acetic anhydride:polysaccharide repeating unit. The material wasincubated with mixing for 2 hours at room temperature. The pH waschecked at the end of 2 hours to verify that is was ˜8.8.

Purification of the Re-N-Acetylated Polysaccharide by TangentialDiafiltration 30 kDa:

To eliminate the particulate formed during the storage, the material isfiltered against a 0.45/0.2 μm filter (Sartobran filter). Note:clarification by centrifugation is also acceptable. The material wasdia-filtered on 30 kDa cellulose membrane (Sartocon Hydrosart 0.1 m2)against 13 volumes of sodium acetate 10 mM with a pressure setting ofΔP[P_(in)−P_(out)]<0.7 bar, TMP[(P_(in)+P_(out))/2]>1.0 (e.g., P_(in)=2bar. P_(out)=1 bar). The material was filter sterilized with a 0.45/0.2μm filter (Sartorius Sartobran filter). The material was stored at 2-8°C. until needed (max 15 days).

Recovery of Polysaccharide:

CaCl₂ 2 M was added to obtain a final concentration of 0.1 M (52.6 mLper 1 L of neutralized mixture) and the suspension was mixed tohomogeneity. Ethanol (96% (v/v)) was added to a final concentration of80% (v/v) (ratio of 4 L per 1 L of solution) and the suspension wasmixed to homogeneity. The precipitate was washed (2-3 times) with freshethanol 96% (˜50 mL each). The precipitate was collected bycentrifugation at 3000×g for 10 min and dried to a powder under vacuum.

Analytical Methods

Wet-Chemical Assays:

The saccharide content was determined by the sialic acid wet-chemicalassay (Svennerholm, L. Biochem. Biophys. Acta 1957, 24, 604). The samplewas hydrolyzed in HCl at 80° C. 90 minutes, neutralized with NaOH andinjected in a DIONEX™ system. Data are processed by CHROMELEON™Software. The saccharides were eluted using a seven minute lineargradient of 90:10 to 60:40 0.1 M NaOH, 0.1 M NaAcetate:0.1 M NaOH, 0.5 MNaNO₃ on a CarboPac PA1 column with PA1 guard at a flow rate of 1.0ml/min.

Free sialic acid was determined by injecting the polysaccharide samplesolubilized in water at 1.0 mg/ml without hydrolyzing the sample. Inthis way it was possible to separate free from bound sialic acid. FIG.29 is an overlay of a polysaccharide sample and standard (gray line) at0.5 μg/ml. In the polysaccharide sample, free sialic acid is notdetected. The peak in the regeneration step was the polysaccharide nothydrolyzed. Free sialic acid is an important parameter because it isrelated with immune response.

The residual protein content was determined by a MicroBCA™ commercialkit (Pierce). The residual nucleic acid content was determined followingthe method published by Sheldon, E. L.; et al. Biochem. Biophys. Res.Comm. 1989, 156(1), 474.

The residual Group B polysaccharide content determined by determiningthe rhamnose residues and using a method based on HPAEC-PAD analysis.Rhamnose is a specific saccharide in the group B carbohydrate that isnot found in the Type polysaccharides and it was used to determine theconcentration of contaminant carbohydrate residue after capsularpolysaccharide purification. The sample assayed was purified GBS typeIII polysaccharide in FIG. 30. The sample did not present a rhamnosepeak indicating the absence of other carbohydrate contaminants. The graychromatogram was obtained by adding rhamnose standard to the sample.Samples and standards were hydrolyzed in TFA 2N at 100° C. for 3.0hours, then evaporated in SpeedVac and reconstituted with 450 μl of H2O.Rhamnose standard curve range is 1.0-10.0 μg/ml. The chromatographicconditions were: a CarboPac PA1 column with PA1 guard with a flow rateof 1.0 ml/min of NaOH 12 mM for 15 minutes followed by 5 minutes ofregeneration with NaOH 500 mM and then re-equilibration in NaOH 12 mMfor 25 minutes.

Chromatographic Analysis:

The approximate molecular weights of the Type polysaccharides wereestimated by HPLC on a SUPEROSE™ 6 HR 10/30 column (GE Healthcare)equilibrated with PBS and calibrated with dextrans.

NMR Analysis:

Samples of purified polysaccharides were prepared by dissolving thepowder in 1 mL of deuterium oxide (D2O, Aldrich) to a uniformconcentration. Aliquots (750 μL) of the samples were transferred to 5-mmNMR tubes (Wilmad). ¹H NMR experiments were recorded at 25° C. on Bruker600 MHz spectrometer, and using 5-mm broadband probe (Bruker). For dataacquisition and processing, XWINNMR software package (Bruker) was used.1-D proton NMR spectra were collected using a standard one-pulseexperiment with 32 scans. The transmitter was set at the HDO frequency(4.79 ppm). ¹H NMR spectra were obtained in quantitative matter using atotal recycle time to ensure a full recovery of each signal (5×Longitudinal Relaxation Time Ti).

2-D homo- and hetero-correlation NMR spectrum were recorded to assignthe 1-D proton NMR profiles (See, FIGS. 25-28). The peak assignment wasalso confirmed by comparison with published data (Michon, F.; Chalifour,R.; Feldman, R.; Wessels, M.; Kasper, D. L.; Gamian, A.; Pozsgay, V.;Jennings. H. J. Infect Immun 1991, 59, 1690 and related papers).

Results and Discussion

This procedure provides a novel simple, fast and effective method forpurifying Type polysaccharides from streptococcal bacteria. It isadvantageous that the process does not involve the use of DNAse, RNAseand protease treatments. The products are recovered in high yields,whereas all the main potential contaminants (proteins, nucleic acids andGroup B polysaccharide) are reduced lower than 1% w/w. The newpurification method can be used for manufacturing of clinical andcommercial materials derived from these capsular polysaccharides.

The product purity was confirmed as reported in Table 24.

TABLE 24 Summary of the product purity of GBS Type Ia, Ib, III and Vpolysaccharides Protein Nucleic Acid Group B Residual Residual PSResidual PS Content¹ Content² content³ Content⁴ (μg/mg (μg/mg (μg/mg(μg/mg PS Type powder) powder) powder) powder) Type Ia 090 935 3 <10 <10Type Ib H36B 757 9 <10 <10 Type III M781 746 1 <10 <10 Type V CJB111 7853 <10 <10 (¹Sialic acid wet-chemical assay; ²MicroBCA protein commercialkit assay; ³Nucleic acid assay; ⁴Group B polysaccharide assay).

Average molecular weights for the Type polysaccharides, estimated bySize Exclusion Chromatography, were ˜200 kDa for the Type Ia, Ib and˜100 kDa for the Type III and V. The structural identity of GBS Type Ia,Ib, III and V polysaccharide was confirmed by 1H NMR spectroscopy (FIGS.25-28).

Example 7 Purification

This example shows a further exemplary purification protocol whichprovides much higher levels of purity than have previously been possiblefor capsular polysaccharides.

Isolation and Purification of GBS Type Ia, 1b, III and V Polysaccharides

Native GBS Type V polysaccharide were extracted and purified frombacteria using the following process steps:

Bacterial Fermentation:

GBS Type V strain (e.g., CJB111) was grown in complex medium. Any methodof culture may be used, although fermentative culture as disclosedherein is preferred.

Inactivation of Fermentation Biomass and Polysaccharide Extraction (BaseTreatment):

If necessary, the biomass may be heated to bring it to room temperature.Sodium hydroxide (4 M) was added to the recovered biomass to a finalconcentration of 0.8 M and mixed to homogeneity. The suspension wassubsequently incubated at 37° C. for 36 hours with mixing.

Neutralization of Biomass:

After extraction with base treatment. TRIS-base 1 M (121.14 g/mol) wasadded to a final concentration of 50 mM (52.6 mL per 1 L of basemixture) and the suspension was mixed to homogeneity. The pH of themixture was adjusted to 7.8 with HCl (6 M) (1:1 dilution of theconcentrated acid).

Alcohol Precipitation:

2 M CaCl₂ was added to a final concentration of 0.1 M (52.6 mL per 1 Lof neutralized mixture) and the suspension was mixed to homogeneity.Ethanol (96% (v/v)) was added to a final concentration of 30% (v/v)ethanol (428 mL per 1 L) and the suspension was mixed to homogeneity.

Tangential Microfiltration:

The supernatant from the alcohol precipitation was recovered by atangential microfiltration on a 0.2 μm cellulose membrane (SartoriusSartocon Hydrosart 0.1 m2) against a dialysis buffer comprising: NaCl(0.5 M)+CaCl₂ (0.1 M)+ethanol 30% (v/v) buffered at pH 7.8. Ten dialysisvolumes were used for the microfiltration. The permeate was filteredusing a 0.45/0.2 μm filter to sterilize the permeate (SartoriusSartobran filter). Note: as an alternative, the retentate can beclarified by centrifugation (retaining the supernatant fluid) and storedat 2-8° C.

Tangential Diafiltration 30 kDa:

To eliminate particulate matter formed during storage, the material wasfiltered with a 0.45/0.2 μm filter (Sartobran filter). The material waspurified by a first diafiltration step using a 30 kDa cellulose membrane(Sartorius Sartocon Hydrosart 0.6 m²) against 20 volumes of TRIS 50 mM,NaCl 0.5 M at pH 8.8 and then against 10 volumes of Na phosphate 10 mMat pH 7.2. Pressure setting: P_(in)=3 bar, P_(out)=1 bar). The retentateof the first diafiltration step was diluted to 10 kg and then treatedwith an acetic acid/sodium acetate solution at pH 4.0 (2 L). Thesuspension obtained from this treatment was filtered using GFPlus 0.45μm capsules (Sartorius) in order to remove precipitate and then filteredonce again using a 0.2 μm membrane filter (Sartobran Sartorius). The pHwas maintained at a value of 4.4±0.1. The filtered product was thendiafiltered again against Na₂CO₃ 0.3M, NaCl 0.3 M at pH 8.8. Afterfurther filtration using a 0.45/0.2 filter, the material was stored at2-8° C. (for a maximum of 15 days) until needed.

Adherent Filtration with CUNO Capsules:

Filtration was carried out using CUNO Z-Carbon R53SLP8 cartridges. Usinga peristaltic pump, the cartridges were assembled in a dedicated holderand then washed with >20.0 L of WFI at a flow rate of 580±40 mL/min. Thecartridges were then washed with >20.0 L of Na₂CO₃ 0.3 M, NaCl 0.3 M atpH 8.8 at the same flow rate. If the volume of the material was lessthan 20 L, then it was diluted to the desired volume with Na₂CO₃ 0.3 M,NaCl 0.3 M buffered at pH 8.8. The material was then filtered andcollected in a sterile bag. The holder was filled with 20 L of Na₂CO₃0.3 M, NaCl 0.3 M at pH 8.8 and filtration conducted to collect 6 L offiltered product. The material was then filtered using a 0.45/0.2 μmfilter. The material was stored at 2-8° C. (for a maximum of 15 days)until needed.

Re-N-Acetylation of Polysaccharide:

Z-Carbon filtered material was treated with an acetic anhydride/ethanolsolution to allow re-N-acetylation. The reactive mixture needed to treat1 L of polysaccharide solution was prepared using the followingproportions: 4.15 mL of acetic anhydride+4.15 mL of ethanol 96%. Thereaction solution was incubated under stirring for 2 hours at roomtemperature. The pH was checked at the end of 2 hours to verify that itwas about 7.

Purification of the Re-N-Acetylated Polysaccharide by TangentialDiafiltration 30 kDa:

The material was diafiltered on 30 kDa cellulose membranes (0.1 m2Sartocon Hydrosart) against 13 volumes of potassium phosphate 10 mM atpH 7.2 with a pressure setting of ΔP[P_(in)−P_(out)]<0.7 bar,TMP[(P_(in)+P_(out))/2]>1.0 (e.g., P_(in)=2 bar. P_(out)=1 bar). Thematerial was then filtered using a 0.45/0.2 μm filter. The material wasstored at −20° C. until needed.

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1. A cultivating medium comprising a Streptococcus strain, a phosphatesource, a carbon source, a vitamin source, and an amino acid source togrow Streptococcus, wherein said vitamin source consists of six or fewervitamins selected from the following list of seven vitamins: biotin,niacinamide, calcium pantothenate, riboflavin, thiamine hydrochloride,pyridoxine hydrochloride and folic acid, wherein two of the vitaminshave to be calcium pantothenate and niacinamide.
 2. The cultivatingmedium of claim 1, wherein said Streptococcus is Streptococcusagalactiae.
 3. The cultivating medium in claim 1, wherein said phosphatesource consists of K₂HPO₄, KH₂PO₄, Na₂HPO₄.H2O, NaH₂PO₄.H2O, or NaCl. 4.The cultivating medium of claim 1, wherein said carbon source isglucose.
 5. The cultivating medium of claim 1, wherein said vitaminsource consists of six or fewer from the following list of sevenvitamins: biotin, niacinamide, calcium pantothenate, riboflavin,thiamine hydrochloride, pyridoxine hydrochloride, and folic acid,wherein two have to be calcium pantothenate and niacinamide.
 6. Thecultivating medium of claim 1, wherein said vitamin source consists offive or fewer from the following list of seven vitamins: biotin,niacinamide, calcium pantothenate, riboflavin, thiamine hydrochloride,pyridoxine hydrochloride, and folic acid, wherein two have to be calciumpantothenate and niacinamide.
 7. The cultivating medium of claim 1,wherein said vitamin source consists of four or fewer from the followinglist of seven vitamins: biotin, niacinamide, calcium pantothenate,riboflavin, thiamine hydrochloride, pyridoxine hydrochloride, and folicacid, wherein two have to be calcium pantothenate and niacinamide. 8.The cultivating medium of claim 1, wherein said vitamin source consistsof three or fewer from the following list of seven vitamins: biotin,niacinamide, calcium pantothenate, riboflavin, thiamine hydrochloride,pyridoxine hydrochloride, and folic acid, wherein two have to be calciumpantothenate and niacinamide.
 9. The cultivating medium of claim 1,wherein said vitamin source consists of calcium pantothenate andniacinamide.
 10. The cultivating medium of claim 1, wherein said aminoacid source consists of nineteen or fewer from the following list ofnineteen amino acids: alanine, arginine, glutamine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, valine, aspartic acid, cysteine hydrochloride,glutamic acid, and tyrosine, wherein fifteen have to be arginine,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, serine, threonine, tryptophan, valine, cysteinehydrochloride, glutamic acid, and tyrosine.
 11. The cultivating mediumof claim 1, wherein said amino acid source consists of eighteen or fewerfrom the following list of nineteen amino acids: alanine, arginine,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, valine, asparticacid, cysteine hydrochloride, glutamic acid, and tyrosine, whereinfifteen have to be arginine, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, serine, threonine, tryptophan,valine, cysteine hydrochloride, glutamic acid, and tyrosine.
 12. Thecultivating medium of claim 1, wherein said amino acid source consistsof seventeen or fewer from the following list of nineteen amino acids:alanine, arginine, glutamine, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, valine, aspartic acid, cysteine hydrochloride, glutamicacid, and tyrosine, wherein fifteen have to be arginine, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,serine, threonine, tryptophan, valine, cysteine hydrochloride, glutamicacid, and tyrosine.
 13. The cultivating medium of claim 1, wherein saidamino acid source consists of sixteen or fewer from the following listof nineteen amino acids: alanine, arginine, glutamine, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, valine, aspartic acid, cysteinehydrochloride, glutamic acid, and tyrosine, wherein fifteen have to bearginine, glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, serine, threonine, tryptophan, valine, cysteinehydrochloride, glutamic acid, and tyrosine.
 14. The cultivating mediumof claim 1, wherein said amino acid source consists of arginine,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, serine, threonine, tryptophan, valine, cysteinehydrochloride, glutamic acid, and tyrosine.
 15. A cultivating mediumcomprising a Streptococcus strain, a yeast extract, a phosphate source,a carbon source, a vitamin source, and optionally an amino acid sourceto grow Streptococcus, wherein said vitamin source consists of four orfewer vitamins selected from the following list of five vitamins:biotin, niacinamide, riboflavin, thiamine hydrochloride and pyridoxinehydrochloride, wherein one of the vitamins has to be biotin.
 16. Thecultivating medium of claim 15, wherein said Streptococcus isStreptococcus agalactiae.
 17. The cultivating medium of claim 15,wherein said vitamin source consists of four or fewer vitamins selectedfrom the following list of five vitamins: biotin, niacinamide,riboflavin, thiamine hydrochloride and pyridoxine hydrochloride, whereinone of the vitamins has to be biotin.
 18. The cultivating medium ofclaim 15, wherein said vitamin source consists of three or fewervitamins selected from the following list of five vitamins: biotin,niacinamide, riboflavin, thiamine hydrochloride and pyridoxinehydrochloride, wherein one of the vitamins has to be biotin.
 19. Thecultivating medium of claim 15, wherein said vitamin source consists oftwo or fewer vitamins selected from the following list of five vitamins:biotin, niacinamide, riboflavin, thiamine hydrochloride and pyridoxinehydrochloride, wherein one of the vitamins has to be biotin.
 20. Thecultivating medium of claim 15, wherein said vitamin source consists ofbiotin.